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Keywords:

  • brain-derived neurotrophic factor;
  • dentate gyrus;
  • nerve growth factor;
  • neurotrophin-3;
  • seizures;
  • temporal lobe epilepsy

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

A significant upregulation of neurotrophins particularly brain-derived neurotrophic factor (BDNF) is believed to be involved in the initiation of epileptogenic changes such as the aberrant axonal sprouting and synaptic reorganization in the injured hippocampus. However, it is unknown which of the neurotrophins are upregulated during the peak period of aberrant mossy fiber sprouting in the chronically injured hippocampus. We measured chronic changes in the levels of BDNF, nerve growth factor (NGF) and neurotrophin-3 (NT-3) in the adult hippocampus using enzyme-linked immunosorbent assay (ELISA) after a unilateral intracerebroventricular administration of kainic acid (KA), a model of temporal lobe epilepsy. For comparison, neurotrophins were also measured from the control intact hippocampus. Further, to see the association between changes in neurotrophin levels and the progression of mossy fiber sprouting, chronic changes in the mossy fiber distribution within the dentate supragranular layer (DSGL) were quantified. In the KA-lesioned hippocampus, the neurotrophins BDNF and NGF were upregulated at 4 days post-lesion, in comparison to their levels in the intact hippocampus. However, the concentration of BDNF reached the baseline level at 45 days post-lesion and dramatically diminished at 120 days post-lesion. In contrast, the upregulation of NGF observed at 4 days post-lesion was sustained at both 45 days and 120 days post-lesion. The concentration of NT-3 was upregulated at 45 days post-lesion but remained comparable to baseline levels at 4 days and 120 days post-lesion. Interestingly, analysis of mossy fiber sprouting revealed that most of the aberrant sprouting in the lesioned hippocampus occurs between 45 days and 120 days post-lesion. Taken together, these results suggest that the period of robust mossy fiber sprouting does not correlate with the phase of post-lesion BDNF upregulation. Rather, it shows a relationship with the time of upregulation of neurotrophins NGF and NT-3.

Abbreviations used
BDNF

brain-derived neurotrophic factor

DSGL

dentate supragranular layer

ELISA

enzyme-linked immunosorbent assay

GABA

gamma-amino-butyric acid

ICV

intracerebroventricular

KA

kainic acid

NGF

nerve growth factor

NT-3

neurotrophin-3

PB

phosphate buffer

PBS

phosphate-buffered saline

PMSF

phenylmethylsulfonyl fluoride

TLE

temporal lobe epilepsy

TMB

tetramethyl benzidine

Intracerebroventricular (ICV) administration of kainic acid (KA) in rat, a model for studying hippocampal lesion recovery and structural and functional changes underlying epileptogenesis, induces selective degeneration of hippocampal CA3 pyramidal neurons and a significant fraction of dentate hilar neurons (Nadler et al. 1980a, 1980b; Shetty and Turner 1996). This pattern of neurodegeneration consistently leads to multiple epileptogenic changes in the hippocampus. The most conspicuous changes comprise a reduction in the number of gamma-amino-butyric acid (GABA) positive interneurons throughout the hippocampus, and sprouting of axons in both CA1 subfield and the dentate gyrus leading to an aberrant reorganization of the circuitry and eventually hyperexcitability (Tauck and Nadler 1985; Wheal 1989; Turner and Wheal 1991; Shetty and Turner 1997, 1999, 2000, 2001; Shetty 2002). As many of these changes closely resemble the changes in the human mesial temporal lobe epilepsy (TLE), ICV administration of KA in rat is widely used as a model for studying TLE (Okazaki et al. 1995; Shetty and Turner 1996). As in the epileptic human hippocampus, following ICV KA-induced lesion, axons of granule cells (mossy fibers) sprout aberrantly into the inner molecular layer of the dentate gyrus, commonly referred to as the dentate supragranular layer (DSGL). Studies using both the surgically removed human TLE specimens and KA-lesioned rat hippocampus reveal that the DSGL mossy fiber sprouting forms new excitatory synaptic connections between granule cells and likely leads to an increased seizure susceptibility in the dentate gyrus (Tauck and Nadler 1985; Cronin and Dudek 1988; Mathern et al. 1993; Mello et al. 1993; Okazaki et al. 1995).

Although the precise reasons for aberrant sprouting of mossy fibers following their target loss (i.e. the loss of CA3 pyramidal neurons and hilar cells) are mostly unknown, it is generally believed that upregulation in neurotrophin signaling following lesions plays a prominent role in the axonal sprouting and aberrant synaptic reorganization in the hippocampus. The neurotrophins brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) have close homology but have distinct effects on the growth and survival of neurons (Leibrock et al. 1989; Hohn et al. 1990). In the hippocampus, all three neurotrophins are profusely expressed and are important for hippocampal plasticity especially pertaining to learning and memory (Ernfors et al. 1990a; Conner et al. 1997). The dentate granule cells, in particular, are well known to co-express BDNF, NGF and NT-3 (Ernfors et al. 1990a; Mathern et al. 1997). Studies utilizing several animal models of epilepsy have revealed that limbic seizures after administration of excitotoxins greatly increase both mRNA and protein levels of neurotrophins, especially the BDNF (Gall and Isackson 1989; Falkenberg et al. 1993; Gall 1993; Lapchak et al. 1993; Cheng and Mattson 1994; Elmer et al. 1998; Rudge et al. 1998; Vezzani et al. 1999). This bestowed the thought that seizure-induced upregulation in neurotrophic factors likely contributes to both structural and functional alterations underlying epileptogenesis (Gall et al. 1991, 1997; Isackson et al. 1991; Lowenstein et al. 1993; Binder et al. 2001). Multiple physiological studies also implicate BDNF for changes underlying the epileptic state in the dentate gyrus after lesions (Kokaia et al. 1995; Gall et al. 1997; Scharfman 1997; Scharfman et al. 1999; Binder et al. 2001). Indeed, intraventricular administration of BDNF receptor (trkB) antibody inhibits the development of kindling, and mossy fibers in the adult rat hippocampus activate trk receptors following seizures (Binder et al. 1999a, 1999b).

Thus, increased levels of neurotrophins could be involved in initiating epileptogenesis in the hippocampus after injury. However, it is unclear whether neurotrophins have a role in the maintenance of chronic epilepsy and/or progression of mossy fiber sprouting, as long-term changes in neurotrophin levels following hippocampal injury (i.e. levels in the chronically injured and epileptic hippocampus) are unknown. Therefore, in this study, we measured changes in the levels of neurotrophins BDNF, NGF and NT-3 in the adult hippocampus following ICV administration of KA at 4, 45, and 120 days post-KA lesion. For comparison, neurotrophins were also measured from the intact hippocampus of control animals and the deafferented hippocampus (i.e. the hippocampus contralateral to the ICV KA administration). We used Emax Immunoassay Systems (Promega, Madison, WI, USA) for measurement of neurotrophic factors BDNF, NGF and NT-3 in this study. Additionally, to see the association between neurotrophin levels and the progression of mossy fiber sprouting, the distribution of mossy fibers in the DSGL was quantified in both control animals and KA treated animals at 45, 120 and 360 days post-lesion using Timm's histochemical staining.

Kainic acid lesions

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

Adult male Fischer 344 rats (Harlan Sprague–Dawley, 4–6 months old) were employed in this study. For this survival surgery, each animal was first injected with an anesthetic cocktail containing ketamine (50 mg/mL), xylazine (6 mg/mL) and acepromazine (0.5 mg/mL) at a dose of 1.25-mL/kg body weight. The head of anesthetized animal was then fixed into the stereotaxic apparatus, and a burr hole was drilled using the following stereotaxic co-ordinates: anteroposterior, 3.7 mm caudal to bregma; lateral, 4.1 mm right lateral to mid line. Following this, a 10-µL Hamilton syringe containing KA solution (0.5 µg of KA/µL of physiological saline) and fitted with a 20-G needle was inserted through the burr hole into the lateral ventricle (i.e. 4.5 mm ventral to the surface of the brain), and 1 µL of KA solution was slowly injected over a period of 10 min. The needle was left in place for an additional 10 min before withdrawal. Following withdrawal of the needle, skin was stapled and animals returned to their cages. All experiments were performed as per the animal protocol approved by the animal studies subcommittee of the Durham Veterans Affairs Medical Center and the Institutional Animal Care and Use Committee of the Duke University Medical Center.

Extraction of neurotrophins

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

Both control animals and KA-lesioned animals (at 4, 45 and 120 days post-lesion) were killed by an overdose of anesthesia, and the brains were rapidly removed from the skull. The entire hippocampus was dissected from the cerebral hemisphere and weighed immediately to get the wet weight of the sample. Each hippocampus was then transferred to an ice-cold lysis buffer containing NaCl (137 mm), Tris–HCL (20 mm), NP40 detergent (1%), glycerol (10%), sodium vanadate (0.5 mm) and the following protease inhibitors (Sigma, St Louis, MO, USA): phenylmethylsulfonyl fluoride (PMSF, 1 mm), aprotinin (10 µg/mL) and leupeptin (1 µg/mL). Following this, every hippocampus was sonicated in 300 µL of ice-cold lysis buffer using Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA, USA) for 10 s. The lysate from each sample was centrifuged at 14 000 g for 15 min at 4°C and the supernatant solutions collected. The supernatant from each sample was diluted five times with Dulbecco's phosphate-buffered saline (PBS) and acidified to pH 2.6. After 15 min of incubation at room temperature, the diluted supernatants were neutralized to pH 7.6, and frozen for subsequent measurements of neurotrophic factors using enzyme-linked immunosorbent assay (ELISA). The above procedure was performed for all samples utilized in this study, as a previous study has indicated that acidification and subsequent neutralization with base increases the amount of detectable neurotrophic factors, particularly BDNF, NGF and NT-3 in extracts of CNS tissues (Okragly and Haak-Frendscho 1997).

ELISA protocol for measurement of neurotrophins

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

The samples from seven different groups were assayed together for each of the three neurotrophins (BDNF, NGF and NT-3) using Emax immunoassay systems from Promega. The groups comprised: (i) intact control hippocampus (n = 5); (ii) hippocampus ipsilateral to KA administration at 4 days post-lesion (n = 5); (iii) hippocampus ipsilateral to KA administration at 45 days post-lesion (n = 5); (iv) hippocampus ipsilateral to KA administration at 120 days post-lesion (n = 5); (v) hippocampus contralateral to KA-administration at 4 days post-lesion (n = 4); (vi) hippocampus contralateral to KA administration at 45 days post-lesion (n = 4); and (vii) hippocampus contralateral to KA administration at 120 days post-lesion (n = 4). The Emax Immunoassay systems used in this study are sensitive for the specific detection of neurotrophins in an antibody sandwich format (Hornbeck 1994). Using this format, BDNF in tissue extracts can be quantified in the range of 15.6–500 pg/mL, NGF in the range of 7.8–500 pg/mL, and NT-3 in the range of 4.7–300 pg/mL.

Flat-bottom 96-well plates (NUNC, Naperville, IL, USA) were first coated with the first primary antibody solution prepared in carbonate-coating buffer (100 µL/well, 1 : 2000 dilution) and incubated for 16 h at 4°C. The first primary antibody was polyclonal in NGF and NT-3 detection kits and monoclonal in BDNF detection kit. Following a wash in TBST (Tris-buffered saline containing Tween-20), the coated wells were incubated with block and sample buffer (1×) for 1 h at room temperature, and washed again with TBST. Standard control samples for each neurotrophic factor were diluted serially (1 : 2) from either 300–0 pg/mL or 500–0 pg/mL and plated to two columns of wells (100 µL/well) designated for standard curve in every plate. The frozen ELISA samples (described above) were thawed on ice, and every sample plated in duplicate for measurement of each of the three neurotrophins. Following 6 h incubation at room temperature, wells were washed in TBST, treated with the second primary antibody solution (1 : 500–1 : 4000) for 2–16 h. The second primary antibody was monoclonal in NGF and NT-3 detection kits and polyclonal in the BDNF detection kit. Then, the wells were washed in TBST, treated with appropriate secondary antibody conjugated to peroxidase for 1–3 h, washed again in TBST and treated with tetramethyl benzidine (TMB) substrate for 10 min. The chromogen reaction was stopped by adding 100 µL of 1 m phosphoric acid. Plates were then read at a wavelength of 450 nm on a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

The data were analyzed using SoftMax computer program associated with the microplate reader. The software program corrects raw data from all unknown samples in relation to the recovery of standard neurotrophic factor samples. The validity of the protocol during every run was confirmed with the optical density values for different known concentrations of the standard, which gave a clear standard curve when the protocol was performed correctly. Values in all samples were normalized per gram of tissue assayed, and the average value for each sample was calculated separately before determining the group means. Then, the amount of BDNF, NGF and NT-3 in different groups was compared using either anova with Student's Newman Keuls multiple comparisons test or the non-parametric Kruskal–Wallis test.

Analysis of KA lesions and the extent of mossy fiber sprouting

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

All animals [normal control (n = 4) and KA-treated animals at 45, 120 and 360 days post-lesion (n = 5–7 at each time-point)] were deeply anesthetized with halothane and perfused transcardially with the modified fixation procedure for Timm's histochemical staining (Mitchell et al. 1993). Briefly, following a rinse in the physiological saline (150 mL, 5–8 min), animals were perfused with 100 mL of 1% sodium sulfide solution followed by 100 mL of 4% paraformaldehyde and finally with a further 50 mL of 1% sodium sulfide. The brains were removed, post-fixed in 4% paraformaldehyde for 3 h at 4°C and cryoprotected in 30% sucrose solution in phosphate buffer (PB). Cryostat sections were cut coronally through the septal hippocampus and collected serially in PB. Twenty-micrometer sections through the septal hippocampus at levels corresponding to 2.8–4.0 mm posterior to bregma (Paxinos and Watson 1986) with a distance of 100 µm between them were selected in each of the animals and processed for Timm's histochemical staining to demonstrate mossy fibers in the hippocampus. This protocol ensured that selected sections were independent from one another to clearly avoid analysis of mossy fiber sprouting from adjacent sections and hence, replication of the findings of the first section.

Timm's histochemical staining was performed as detailed elsewhere (Danscher 1981). Briefly, 8–10 sections from the septal hippocampus of every rat with a distance of 100 µm between them were mounted on gelatin-coated slides and air-dried. Then, the slides containing sections were rinsed in distilled water, placed in jars, covered with the developer for Timm's staining, and immediately placed in the water bath at 26°C for 150 min. The jars were placed in a dark room to reduce the autocatalytic staining of the solution. After development, the sections were rinsed several times in distilled water, processed for light Nissl staining with cresyl violet, dehydrated, cleared, and coverslipped with permount. Additional adjacent sections were stained for Nissl alone for comparison. Nissl staining demonstrated hippocampal cytoarchitecture in both control and lesioned animals and, in addition, confirmed the pattern of the hippocampal lesion in all animals administered with KA. The pattern of lesion in the septal hippocampus due to the unilateral ICV KA administration was consistent in a vast majority (> 90%) of animals. The lesion was characterized by: (i) a complete loss of CA3 pyramidal cells throughout the septal hippocampus ipsilateral to the KA administration except for a tiny region of CA3 adjacent to CA2 cell layer where larger pyramidal cells of CA3a were preserved; (ii) sparing of CA1 pyramidal cells and dentate granule cells ipsilateral to the KA administration; and (iii) completely intact principal cell layers in the septal hippocampus contralateral to the KA administration. For morphometric analysis of mossy fiber sprouting into the DSGL following ICV KA administration, in every lesioned group, sections were analyzed from only those animals that demonstrated the above three lesion characteristics and also exhibited dense staining of mossy fibers throughout the dentate hilus.

Morphometric analysis of mossy fiber sprouting into the DSGL

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

KA lesion induced mossy fiber sprouting into the DSGL was compared between different groups of animals using two separate and independent quantitative methods. These included measurement of the average width of mossy fiber sprouting and the density of Timm's granules in the dentate inner molecular layer. The degree of sprouting was measured in all regions of the dentate gyrus (supra- and infra-pyramidal blades and the crest) with both quantitative methods. In each animal, four sections through the septal hippocampus (separated by 100 µm distance) were measured. Measurements in sections from various groups were performed in a blinded fashion using experimental codes. The average width of DSGL sprouting was measured using Scion Image (Scion Corporation, Frederick, MD, USA) based on NIH Image for McIntosh. The length and area of supragranular mossy fiber sprouting in all three regions of the dentate gyrus were marked in every section, and the average width of DSGL sprouting for each relevant region of the dentate gyrus was calculated for every animal before determining the group means and standard errors between animals.

The densitometric analysis consisted of measuring the optical density of the inner and outer molecular layer in these representative sections (four per animal) using a compound microscope under a 40X objective. Using a digital camera (Dage MTI), a representative area of the supra- and infra-pyramidal blades and also the dentate crest was imaged, digitized and stored. Then, the Scion Image was used to draw a region of interest in both the supragranular region (inner molecular layer) and the middle and outer molecular layer on the same image. The dimensions of these regions were 70 × 70 µm, with an area of approximately 4.9 × 103µm2 for each region. The background of the inner molecular layer was very similar to that of the middle and outer molecular layers, if no supragranular sprouting was present. Thus, it was assumed that the more distal molecular layer was a control region for the inner molecular layer, and the difference in pixel value between inner and outer molecular layers was determined for the corresponding regions of interest. This measurement provided a highly objective and specific measure of the density of Timm's granule staining in the DSGL, corrected for the background control density expected in the molecular layer and other non-specific differences between sections, such as background staining level. These values were expressed in final form as a difference (inner molecular layer minus the outer molecular layer value), which ranged between no difference (0, with no sprouting) and 255, for extremely dense Timm's staining representative of sprouting.

Four sections (20 µm thick) were analyzed in each animal for all measurements. Quantification was performed using the following four groups of animals: (a) intact control hippocampus (n = 4); (b) hippocampus ipsilateral to KA administration at 45 days post-lesion (n = 4); (c) hippocampus ipsilateral to KA administration at 120 days post-lesion (n = 4); and (d) hippocampus ipsilateral to KA administration at 360 days post-lesion (n = 4). All animals measured in the KA-lesioned groups have satisfied the selection criteria described earlier. The mean value for each of the three regions of dentate gyrus (supra- and infra-pyramidal blades and the crest) was calculated separately for each animal by using data from four sections before the means and standard errors were determined for the total number of animals included per group. Mean values between different age groups of animals were separately compared for every region of the dentate gyrus. Statistical comparisons involved anova with Student–Newman–Keuls multiple comparisons test. To determine mean sprouting values for the entire dentate gyrus in terms of both width of sprouted area and density of sprouted terminals in the DSGL, individual values measured from supra- and infra-pyramidal blades and the crest of the dentate gyrus were averaged in every animal, and then, means and standard errors were calculated in every group for the total number of animals included for measurement. The mean values for both width of sprouted area and density of sprouted terminals were compared statistically between different groups of animals.

Behavioral changes in animals after intracerebroventricular KA administration

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

Behavior of animals was monitored for 4 h after ICV administration of KA. None of the KA-administered animals showed behavioral changes indicative of seizure activity, as commonly seen after either systemic or local intracerebral injections of KA (Sperk 1994). These findings are consistent with the earlier findings that ICV KA administration, particularly at the low dose used in this study, does not produce behavioral seizure activity but consistently destroys CA3 pyramidal and hilar cells of the hippocampus (Nadler et al. 1978; Sperk 1994; Shetty and Turner 1995a, 1995b). However, electrographic hyperactivity may occur in the hippocampus after the lesion and could contribute to cell damage, but this was not measured in this study.

Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

The average concentration of BDNF in the intact adult hippocampus was 30.7 ng/g wet weight of the hippocampus. Following ICV KA administration, the concentration of BDNF in the hippocampus ipsilateral to KA administration (i.e. the CA3-lesioned hippocampus) increased significantly (Fig. 1). At an early post-lesion delay of 4 days, the average concentration of BDNF in the CA3-lesioned hippocampus was 54.5 ng/g wet weight, which is equivalent to 179% of BDNF level in the intact control hippocampus (p < 0.001; Fig. 1). However, the concentration of BDNF decreased with time after the lesion. At a delayed post-lesion time-point of 45 days, the average BDNF concentration in the CA3-lesioned hippocampus was 30.1 ng/g wet weight, which is 98% of BDNF level in the intact control hippocampus (p > 0.05; Fig. 1). Further evaluation of the BDNF concentration at an extended post-lesion period of 120 days revealed a dramatically diminished level of BDNF in the chronically lesioned hippocampus. At 120 days post-lesion, the average concentration of BDNF was 11.2 ng/g-wet weight, which is equivalent to 37% of BDNF level in the intact control hippocampus (p < 0.01; Fig. 1). Thus, following a robust increase at early post-lesion, the level of BDNF in the CA3-lesioned hippocampus drops to the baseline level within 45 days of the ICV KA lesion, and undergoes a dramatic decrease by 120 days post-lesion. In hippocampus contralateral to the ICV KA administration, there was an insignificant increase in the BDNF level at 4 days post-lesion (38.1 ng/g wet weight; 124% of control, p > 0.05; Fig. 1). However, the level of BDNF declined steadily (20.3 ng/g wet weight at 45 days post-lesion, 66% of control, p > 0.05) and reached levels that are significantly less than the control intact hippocampus at 120 days post-lesion (13.4 ng/g wet weight; 44% of control, p < 0.05; Fig. 1). Thus, the hippocampus contralateral to the KA administration (i.e. the deafferented hippocampus) exhibits a significantly reduced level of BDNF at 120 days post-lesion, despite having no apparent cell loss.

image

Figure 1. Alterations in the brain-derived neurotrophic factor (BDNF) content of the hippocampus following unilateral intracerebroventricular kainic acid (KA) administration. In comparison to the intact control hippocampus, the hippocampus ipsilateral to the KA administration (CA3-lesioned hippocampus) exhibits a significant upregulation at 4 days post-lesion, baseline levels at 45 days post-lesion, and a significant downregulation at 120 days post-lesion. Whereas, the hippocampus contralateral to the KA administration (deafferented hippocampus), exhibits BDNF levels that are closer to baseline at 4 and 45 days post-lesion but significantly lower at 120 days post-lesion.

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Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

The intact adult hippocampus exhibited an average concentration of 27.3 ng of NGF/g wet weight of the hippocampus. After ICV KA administration, the concentration of NGF in the hippocampus ipsilateral to KA administration increased significantly and maintained at that level until 120 days post-lesion (Fig. 2). The average concentration of NGF in the CA3-lesioned hippocampus was 46.2 ng/g wet weight at 4-days post-lesion (169% of intact control hippocampus, p < 0.001), 46.9 ng/g wet weight at 45 days post-lesion (172% of intact control, p < 0.001), and 40.7 ng/g wet weight at 120 days post-lesion (149% of intact control, p < 0.001; Fig. 2). Thus, the adult hippocampus demonstrates a persistently increased level of NGF following KA-induced CA3 lesion. In hippocampus contralateral to the ICV KA administration, NGF concentration exhibited a significant increase at 45 days post-lesion (39.3 ng/g wet weight; 144% of control, p < 0.01). However, at both early (4 days) and late (120 days) post-lesion time-points, the NGF level remained comparable to the intact control level (p > 0.05). Thus, the deafferented hippocampus exhibits NGF levels that are mostly comparable to the intact control hippocampus except for a transient increase at 45 days post-lesion.

image

Figure 2. Changes in the levels of nerve growth factor (NGF) in the hippocampus after unilateral intracerebroventricular kainic acid (KA) administration. In comparison to the intact control hippocampus, the hippocampus ipsilateral to the KA administration (CA3-lesioned hippocampus) exhibits a significant upregulation at all post-lesion time-points. In contrast, the hippocampus contralateral to the KA administration (deafferented hippocampus) exhibits NGF levels that are closer to baseline at 4 and 120 days post-lesion but significantly higher at 45 days post-lesion, implying a delayed and transient upregulation of NGF following deafferentation.

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Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

In the intact adult hippocampus, the average concentration of NT-3 was 14.0 ng/g wet weight of the hippocampus. Following ICV KA administration, the concentration of NT-3 in the hippocampus ipsilateral to KA administration increased steadily. At 4 days post-lesion, the average concentration of NT-3 in the CA3-lesioned hippocampus increased to 17.5 ng/g wet weight of the hippocampus (125% of intact control hippocampus) but this increase was not significant statistically (p > 0.05; Fig. 3). At 45 days post-lesion, the average NT-3 concentration in the CA3-lesioned hippocampus increased to 23.4 ng/g wet weight, which is equivalent to 167% of NT-3 level in the intact control hippocampus (p > 0.001; Fig. 3). Interestingly, the concentration of NT-3 at 120 days post-lesion (15.6 ng/g wet weight) was highly comparable to the level observed at 4 days post-lesion and was not significantly different from the intact control hippocampus (Fig. 3). Thus, the upregulation of NT-3 following ICV KA induced CA3 lesion progresses steadily and becomes significant at 45 days post-lesion. Further, unlike BDNF, NT-3 concentration in the chronically lesioned hippocampus remains closer to the intact control hippocampus. In hippocampus contralateral to the ICV KA administration, NT-3 concentration remained comparable to the intact control hippocampus at all post-lesion time-points (Fig. 3), suggesting that the deafferented hippocampus exhibits no changes in the concentration of NT-3.

image

Figure 3. Alterations in the levels of neurotrophin-3 (NT-3) in the hippocampus after unilateral intracerebroventricular kainic acid (KA) administration. In relation to the intact control hippocampus, the hippocampus ipsilateral to the KA administration (CA3-lesioned hippocampus) exhibits no change at 4 days and 120 days post-lesion but significant upregulation at 45 days post-lesion, suggesting that NT-3 production increases in a delayed fashion after CA3 lesion. In the hippocampus contralateral to the KA administration (deafferented hippocampus), NT-3 levels remain comparable to the intact control hippocampus at all post-KA time-points.

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Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

Unilateral ICV KA administration produced degeneration of ipsilateral CA3 pyramidal neurons, consistent with previous reports (Nadler et al. 1978, 1980a, 1980b; Shetty and Turner 1995a, 1995b, 1996, 1997). In this study, we analyzed the lesion size by examining 20-µm serial sections through the entire length of the septal hippocampus. This analysis demonstrated that, in a great majority of animals (> 90%), CA3 pyramidal cells were completely lost except for a tiny region of the CA3 adjacent to CA2 where larger pyramidal cells of CA3a were preserved. However, the CA1 pyramidal and dentate granule cell layers were spared on the side of KA administration at all post-lesion time-points (Fig. 4). In addition, in hippocampus contralateral to the KA administration, all principal cell layers were clearly intact (Fig. 4).

image

Figure 4. Hippocampal cytoarchitecture in control (untreated) and kainic acid (KA)-treated rats. (a) Hippocampus from a control rat. (a1, a3, and a5) Hippocampus ipsilateral to the KA administration at 4 days (a1), 45 days (a3) and 120 days (a5) post-KA administration. (a2, a4, and a6) Hippocampus contralateral to the KA administration at 4 days (a2), 45 days (a4), and 120 days (a6) post-KA administration. A clear loss of CA3 pyramidal neurons is evident in the hippocampus ipsilateral to the KA administration at all post-lesion time-points (a1, a3, a5). Asterisks denote the degenerated CA3 cell layer. In hippocampus contralateral to the KA administration (a2, a4, a6), all hippocampal cell layers (including CA3 pyramidal cell layer) remain intact at all post-KA time-points. DG, dentate gyrus. Scale bar, a = 200 µm. a1–a6 = 400 µm.

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Mossy fiber distribution in the intact control hippocampus

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

Mossy fiber distribution was studied using Timm's histochemical staining, which selectively identifies zinc within mossy fibers in the hippocampus. Darkly stained mossy fibers in both dentate hilus and the stratum lucidum of the CA3 region were observed with this method (Figs 5a1 and a2). A very sparse distribution of Timm's granules indicating mossy fiber terminals was occasionally present in certain regions of the DSGL.

image

Figure 5. The hippocampus ipsilateral to the KA administration (i.e. CA3-lesioned hippocampus) exhibits a highly robust mossy fiber sprouting into the dentate supragranular layer (DSGL). (a1) The distribution of mossy fibers in the intact control hippocampus. Note the marked absence of mossy fiber terminals in the DSGL. (b1) The sprouted mossy fiber terminals in the DSGL of the CA3-lesioned hippocampus at 45 days post-KA administration. (c1) A dense broad band of sprouted mossy fibers in the DSGL of the CA3-leisoned hippocampus at 120 days post-KA administration. (d1) The degree of DSGL sprouting in the CA3-lesioned hippocampus at 360 days post-KA administration. (a2, b2, c2, and d2, respectively) Magnified view of the upper blade of the dentate gyrus from a1, b1, c1, and d1. Note that the extent of mossy fiber sprouting at 360 days post-lesion (d1, d2) remains comparable to the degree of sprouting observed at 120 days post-lesion (c1, c2). DH, dentate hilus; GCL, granule cell layer; SL, stratum lucidum. Scale bar, a1, b1, c1, and d1, 400 µm; a2, b2, c2 and d2, 100 µm.

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Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

Mossy fiber sprouting into the DSGL was examined at 45, 120 and 360 days post-KA administration (Fig. 5). The reliability of Timm's histochemical method in detecting the sprouted mossy fiber collaterals in the DSGL has been well demonstrated by correlation with tracing of mossy fibers using biocytin injections and electron microscopy (Okazaki et al. 1995). The CA3-lesioned hippocampus (hippocampus ipsilateral to ICV KA administration) exhibited a highly robust mossy fiber sprouting into the DSGL. The mossy fiber terminals were clearly seen in the DSGL at 45 days post-lesion (Figs 5b1 and b2). The pattern of sprouting was mostly patchy at this time with maximal sprouting in the ends of upper and lower blades. However, by 120 days post-lesion, a dense broad band of sprouted mossy fibers filled the DSGL (Figs 5c1 and c2). The pattern of sprouting at this time appeared more uniform throughout the dentate gyrus. Interestingly, the extent of mossy fiber sprouting at 360 days post-lesion appeared similar to the extent of sprouting observed at 120 days post-lesion (Figs 5d1 and d2).

Quantification of DSGL mossy fiber sprouting in the CA3-lesioned hippocampus revealed a highly significant increase of Timm's granules in the DSGL of all three regions of the dentate gyrus at all post-lesion time points, compared to the intact control hippocampus (Fig. 6). The average width of mossy fiber sprouting into the DSGL varied in different regions of dentate gyrus (Fig. 6a). Comparison of the width of sprouted zone in different regions of the dentate gyrus between different post-lesion time-points revealed a dramatic increase in mossy fiber sprouting into the DSGL between 45 days post-lesion and 120 days post-lesion. The increase in mossy fiber sprouting during this period was 320% in the upper blade (p < 0.001), 338% in the lower blade (p < 0.01), 284% in the crest (p < 0.01), and 316% in the entire dentate gyrus (p < 0.001; Fig. 6a). In contrast, there was no significant increase in the width of sprouted zone after 120 days post-lesion, as the width of sprouting at 360 days post-lesion in different regions of the dentate gyrus was comparable to that observed at 120 days post-lesion (p > 0.05; Fig. 6a).

image

Figure 6. Histograms illustrate the extent of aberrant mossy fiber sprouting into the DSGL of the CA3-lesioned hippocampus at 45 days, 120 days and 360 days post-KA administration. (a) shows the average width of sprouted area in the dentate supragranular layer. UB, upper blade of the dentate gyrus; LB, lower blade of the dentate gyrus. Note that the width of sprouted area increases dramatically between 45 days post-lesion and 120 days post-lesion in all regions of the dentate gyrus. (b) The average density of sprouted mossy fiber axons in the dentate supragranular layer. Note that, like the width of sprouted area, the density of mossy fiber terminals increases dramatically between 45 days post-lesion and 120 days post-lesion. Further, both width and density measurements show that the extent of sprouting does not increase significantly after 120 days post-lesion implying that the maximal extent of DSGL mossy fiber sprouting occurs by 4 months post-lesion and is maintained at the same level thereafter.

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Analysis of the density of the sprouted area confirmed a similar trend (Fig. 6b). The increase in density of mossy fiber terminals in the DSGL between 45 days post-lesion and 120 days post-lesion was 288% in the upper blade, 202% in the lower blade, 263% in the crest, and 244% in the entire dentate gyrus (p < 0.001; Fig. 6b). Thus, following ICV KA induced CA3 lesion, the hippocampus exhibits a highly robust mossy fiber sprouting into the DSGL. The maximal extent of DSGL mossy fiber sprouting occurs between 45 days and 120 days post-lesion, and after 120 days post-lesion the degree of sprouting is maintained at the same level until 360 days post-lesion, the longest time-point analyzed in this study. Additionally, the differences observed in this study between normal untreated rats and ICV KA-administered rats are due to chronic effects of KA-induced hippocampal lesion and not due to the surgical procedure. This is because our earlier studies have shown that both hippocampal cytoarchitecture and mossy fiber distribution remain comparable between rats treated with ICV vehicle solution (i.e. sham surgery) and untreated rats (Shetty and Turner 1995a).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

Using a kainate model of temporal lobe epilepsy in rat, this study investigated whether the phase of upregulation of specific neurotrophins after hippocampal injury positively correlates with the post-lesion period of robust aberrant dentate mossy fiber sprouting. The results, for the first time, provide evidence that the post-lesion period of BDNF protein upregulation in the hippocampus does not show a relationship with the post-lesion time-course of robust aberrant dentate mossy fiber sprouting. Instead, these results emphasize that the period of robust aberrant mossy fiber sprouting following hippocampal injury parallels the time of upregulation of NGF and NT-3.

Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

The levels of neurotrophins BDNF and NGF were clearly elevated at an early post-lesion delay of 4 days in the CA3-lesioned hippocampus, in comparison to their levels in the intact control hippocampus. The increase in concentration at this time was 79% for BDNF and 69% for NGF. These increases in BDNF and NGF proteins at early post-lesion are consistent with the earlier mRNA studies in several animal models of epilepsy, which showed that surviving hippocampal cells particularly granule cells dramatically increase their levels of mRNAs for BDNF and NGF after glutamate receptor activation and neuronal injury (Ballarin et al. 1991; Ernfors et al. 1991; Gall et al. 1991; Isackson et al. 1991; Dugich-Djordjevic et al. 1992; Rocamora et al. 1992; Gall 1993; Lauterborn et al. 1994). However, the concentration of NT-3 protein at 4 days post-lesion, though exhibited 25% increase, remained statistically comparable to that of the intact control hippocampus. This observation differs considerably from some of the earlier NT-3 mRNA studies in the injured hippocampus showing about 80% decrease in NT-3 mRNA levels in granule cells after acute hippocampal stimulation or injury (Ernfors et al. 1991; Lindvall et al. 1992; Takedo et al. 1992; Gall 1993). The discrepancy is likely due to differences in the post-lesion delay at the time of analysis because, the above studies analyzed NT-3 mRNA at very early time points (0–24 h) after the injury; whereas, in this study, the NT-3 protein was quantified at 4 days after the hippocampal injury. Indeed, NT-3 mRNA studies performed at 2 and 4 days post-injury report increased levels of NT-3 mRNA (Rocamora et al. 1993; Takedo et al. 1993). Further, unlike earlier NT-3 mRNA studies where quantification was restricted to the dentate gyrus, this study measured NT-3 protein concentration from the entire lesioned hippocampus and, hence, some of the measured NT-3 protein in this study could be from reactive glial cells and degenerating neurons (Rocamora et al. 1993).

Thus, of the three neurotrophins analyzed in this study, the concentrations of BDNF and NGF are clearly upregulated at 4 days post-lesion in the CA3-lesioned hippocampus. The neurotrophins BDNF and NGF regulate neuronal morphology, plasticity, and synaptogenesis in many regions of the CNS (Barde 1989; Binder et al. 2001). The neurotrophin BDNF in particular can enhance excitatory synaptic transmission (Lohof et al. 1993; Kang and Schuman 1995), reduce inhibitory synaptic transmission (Tanaka et al. 1997), and modulate synaptic efficacy in the hippocampus (Figurov et al. 1996; Korte et al. 1996; Patterson et al. 1996). In fact, the transgenic mice that overexpress BDNF protein have more severe seizures in response to the excitotoxin KA (Croll et al. 1999). Furthermore, the development of kindling is inhibited in BDNF heterozygous (+/–) mice with reduced BDNF protein synthesis (Kokaia et al. 1995), following selective blockade of BDNF (trkB) receptors (Binder et al. 1999a), and after intraventricular infusion of anti-NGF antisera (Van der Zee et al. 1995). In the same vein, NGF infusion enhances the development of kindling and kindling induced axonal growth (Adams et al. 1997). Additionally, the phosphorylated form of trk receptors and BDNF immunoreactivity demonstrate a clear increase after KA-induced seizures (Binder et al. 1999b). Taken together, the above observations suggest a role for BDNF and NGF in hippocampal hyperexcitability after injury.

Aberrant sprouting of mossy fibers into the DSGL, a morphological change that occurs after injury or seizures in the hippocampus, is also linked to hyperexcitability in the dentate gyrus of the injured hippocampus (Tauck and Nadler 1985; Okazaki et al. 1995). Studies show that the degree of DSGL sprouting after hippocampal injury correlates with both antidromically evoked burst firing and development of spontaneous seizures, which normally do not arise from the dentate gyrus of the intact hippocampus (Cronin and Dudek 1988; Mathern et al. 1993; Mello et al. 1993; Okazaki et al. 1995). Although the progression of aberrant mossy fiber sprouting is slow and usually takes about 4 months to show the maximal level of sprouting, the initiation of this sprouting occurs soon after the hippocampal injury. However, our earlier study, analyzing mossy fiber sprouting at 4 and 11 days post-KA lesion (i.e. during the peak period of neurotrophic activity after KA-lesion), has shown that initial sprouting of mossy fibers is restricted to the dentate hilus and the inner regions of the dentate granule cell layer (Shetty and Turner 1995b). In the latter time-course study, during the first month after KA-lesion, it was apparent that the growth of mossy fibers into the DSGL occurs after the dentate hilus gets filled up with the sprouted mossy fibers, and a patchy distribution of Timm's positive mossy fibers is obvious in the DSGL by about 26 days post-KA lesion (Shetty and Turner 1995b). Thus, the initiation of mossy fiber sprouting occurs soon after the hippocampal injury though the spread of sprouted axons into the DSGL takes some time after KA lesion. Therefore, it is possible that the pathological upregulation of BDNF and NGF proteins at early post-lesion, as observed in this and an earlier study (Lowenstein et al. 1993), play a role in the initiation of aberrant dentate mossy fiber sprouting after the hippocampal injury. Indeed, at early post-lesion, trkB immunostaining exhibits a clear increase in regions where mossy fibers are present (Katoh-Semba et al. 1999). From this, it appears that the BDNF released from mossy fibers immediately after KA-induced degeneration of CA3 pyramidal neurons and dentate hilar cells induces morphological changes in granule cells by mediating signals to the cells by binding to trkB at cell surfaces. Thus, a newly synthesized, anterogradely transported, and secreted BDNF likely influences the initial sprouting of axons from granule cells (i.e. mossy fibers). Indeed, a recent study suggests that intrahippocampal infusion of BDNF into intact adult rats initiates mossy fiber sprouting and seizure activity (Scharfman et al. 2002).

Association between neurotrophin levels and the period of robust mossy fiber sprouting

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

The concentration of BDNF reaches the intact control level by 45 days post-lesion, and declines at 120 days post-lesion to a level that is significantly lower than the intact control hippocampus. On the contrary, the extent of upregulation of NGF observed at 4 days post-lesion persists until 120 days post-lesion. Further, the concentration of NT-3 shows significant upregulation at 45 days post-lesion but reaches the concentration seen in the intact control hippocampus by 120 days post-lesion. Interestingly, evaluation of the time-course of progression of aberrant mossy fiber sprouting into the DSGL in the TLE model used in this study reveals very robust sprouting between 45 days and 120 days post-lesion. The above results imply that the post-lesion period of BDNF upregulation in the hippocampus does not correlate with the post-lesion time-course of robust aberrant mossy fiber sprouting. On the contrary, the results support that the period of robust aberrant mossy fiber sprouting following hippocampal injury parallels the period of upregulation of NGF and NT-3. Although it is premature to conclude a clear ‘cause and effect’ relationship between NGF and NT-3 levels and robust mossy fiber sprouting, the present results suggest that, in the lesioned hippocampus (or after limbic seizures), the neurotrophins NGF and NT-3 have significantly greater role than BDNF in the progression of mossy fiber sprouting with time after lesion. This is because both NGF and NT-3 are upregulated and BDNF is downregulated during the period of robust mossy fiber sprouting. Enhanced sprouting of mossy fibers in the BDNF +/–mice (in comparison to wild-type mice) following kindling despite having very low levels of BDNF (Kokaia et al. 1995), and failure to inhibit KA-induced mossy fiber sprouting with blocking of BDNF signaling via trkB receptor antibody (trkB-Fc) in hippocampal explant cultures (Routbort et al. 1997) also support the above conclusion. Additionally, an intraventricular infusion of NGF following kindling increases DSGL mossy fiber sprouting (Adams et al. 1997). Interestingly, the mRNA studies in sclerotic hippocampi of TLE patients suggest a positive correlation between robust aberrant mossy fiber sprouting and increased expression of all three neurotrophins BDNF, NGF, and NT-3 in the dentate gyrus (Mathern et al. 1997). However, the extent of upregulation relative to the intact control hippocampus was not determined directly in the latter study, as the values from surgically removed hippocampi of TLE patients were compared with hippocampi from autopsies within a few hours after death and hippocampi of patients having mass lesions outside the hippocampus. The discrepancy in BDNF expression between the chronic animal model of TLE used in this study and the human TLE specimens with hippocampal sclerosis could also be due to differential loss of neurons in the hippocampus. This is because the animal model used in this study has major cell loss in the CA3 subfield and the dentate hilus, whereas the human TLE specimens had severe cell loss in the CA1 region and the dentate hilus (Mathern et al. 1997). Additionally, it is possible that an increased BDNF mRNA expression in chronic epilepsy does not translate into an increased BDNF protein production.

Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

Like BDNF and its receptor trkB, NT-3, and its receptor trkC are found in brain regions associated with epilepsy and synaptic plasticity. This includes the various components of the hippocampal formation and the amygdala (Ernfors et al. 1990b; Lamballe et al. 1991; Merlio et al. 1992; Bengzon et al. 1993; Xu et al. 2002), suggesting a role for NT-3 in neuronal plasticity in the adult brain. However, mRNA for NT-3 remains unchanged or undergoes a decrease in hippocampal neurons following seizure activities. This is in sharp contrast to increased NGF and BDNF mRNA observed in hippocampal and neocortical neurons after seizures (Ernfors et al. 1991; Rocamora et al. 1992; Bengzon et al. 1993; Kokaia et al. 1996). Thus, the role for NT-3 in epileptogenesis appears different from BDNF and NGF.

A recent study demonstrates that continuous infusion of NT-3 in the absence of electrical activation results in sprouting of mossy fibers into the DSGL and stratum oriens of the CA3 region (Xu et al. 2002). However, continuous NT-3 infusion in kindled animals retards behavioral seizures and inhibits kindling-induced mossy fiber sprouting into the DSGL (Xu et al. 2002). When taken together with the present results showing that the period of robust mossy fiber sprouting correlates with the phase of moderately increased levels of NT-3, it is plausible that a moderate increase in NT-3 promotes DSGL mossy fiber sprouting and a greater increase in NT-3 inhibits mossy fiber sprouting. The inhibitory effect of NT-3 on mossy fiber sprouting likely involves down-regulation of the high-affinity trkA and trkC receptors and attenuation of trk phosphorylation, which in turn leads to a loss of responsiveness to NGF and NT-3 (Xu et al. 2002). Thus, NT-3 can exert opposing effects on mossy fiber sprouting, depending on its overall level. In the present study it is likely that a moderate increase in NT-3 observed after KA lesion promoted mossy fiber sprouting, as this moderate increase might not be adequate to induce downregulation of trk A and trkC receptors. This is in contrast to the scenario in kindled animals receiving continuous infusion of exogenous NT-3, where the overall NT-3 levels might have increased greatly (because of both infusion of exogenous NT-3 and the potential kindling induced upregulation of endogenous NT-3) and lead to downregulation of trkA and trkC receptors.

Regarding NGF, the findings in both previous studies and this study strongly support the hypothesis that NGF plays a major role in the progression of aberrant mossy fiber sprouting into the DSGL after hippocampal injury or seizures (van der Zee et al. 1995; Adams et al. 1997). Nevertheless, the mechanism by which NGF promotes epileptogenesis is not clear. In view of the previous studies suggesting that NGF receptors (both trkA and p75) are minimally expressed in dentate granule cells (Barker-Gibb et al. 2001), it is intriguing that NGF drives granule cells into sprouting their axons in the wrong direction. It may be that the expression of trkA and p75 receptors increases in the dentate granule cells of the lesioned hippocampus (due to KA-induced target loss and deafferentation) and this in turn facilitates the effect of NGF on mossy fiber sprouting. Alternatively, it is possible that the effect of NGF on mossy fiber sprouting is mediated through local astrocytes expressing trkA (Barker-Gibb et al. 2001; McCarthy et al. 2002). NGF may suppress inhibitory molecules on astrocytes and thereby promote mossy fiber sprouting. Additionally, septal afferents rich in trkA receptors could also be involved. This may involve transport of NGF into the septal neurons and the septal neurons responding by sending critical signals to granule cells.

Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References

The downregulation of BDNF could represent an adaptive mechanism in the injured hippocampus to minimize seizures in the chronically lesioned hippocampus, as the reorganized circuitry is already seizure-prone (Tauck and Nadler 1985; Okazaki et al. 1995). This is consistent with the downregulation of dentate granule cell calbindin observed in the chronically lesioned hippocampus (Nagerl et al. 2000; Shetty 2001). Alternatively, the reduced synthesis of BDNF in the chronically lesioned hippocampus could be related to the loss of target cells (i.e. CA3 pyramidal cells and dentate hilar cells) experienced by the dentate granule cells. This is because, in the intact hippocampus, BDNF synthesized by dentate granule cells gets transported anterogradely into the CA3 region and released at the mossy fiber/CA3 synapses (Smith et al. 1997; Binder et al. 2001).

Based on the excitatory effects of BDNF in normal mature animals, it has been speculated that BDNF upregulation in the adult brain could predispose certain areas to seizures or even cause seizures (Binder et al. 2001). Studies have shown that acute application of BDNF to hippocampal slices obtained from intact control rats enhances the efficacy of excitatory mossy fiber synapse on to CA3 pyramidal cells (Scharfman 1997). In hippocampal slices isolated from pilocarpine-treated rats (another model of TLE), BDNF enhances responses to stimulation of mossy fiber collaterals in the inner molecular layer (Scharfman et al. 1999). Moreover, BDNF exposure in these epileptic animals leads to seizure like events, suggesting that BDNF is likely more potent after seizure- or injury-induced mossy fiber sprouting. Thus, significant BDNF upregulation in injured animals exhibiting spontaneous seizures can considerably increase the frequency and the severity of seizures. However, the results of the current study suggest that BDNF is significantly downregulated in the chronically lesioned hippocampus. In the context of the above actions of BDNF, the reduced concentration of BDNF in the chronically lesioned hippocampus appears beneficial and could actually reduce the frequency of seizures. However, a severe BDNF downregulation can also have some adverse effects on the epileptic hippocampus because, BDNF is known to increase neuropeptide Y (NPY) concentration (Croll et al. 1994) and increased NPY concentration can reduce seizure generation (Baraban et al. 1997). Thus, the frequency and severity of seizures in the chronically lesioned hippocampus may be regulated by the overall concentration of BDNF in the hippocampus, as BDNF can have epileptogenic effects (Scharfman et al. 1999) as well as anticonvulsant effects (Croll et al. 1994).

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kainic acid lesions
  5. Extraction of neurotrophins
  6. ELISA protocol for measurement of neurotrophins
  7. Analysis of KA lesions and the extent of mossy fiber sprouting
  8. Morphometric analysis of mossy fiber sprouting into the DSGL
  9. Results
  10. Behavioral changes in animals after intracerebroventricular KA administration
  11. Changes in BDNF levels in the adult hippocampus following unilateral ICV KA administration
  12. Changes in NGF levels in the adult hippocampus following unilateral ICV KA administration
  13. Alterations in NT-3 concentration in the adult hippocampus after unilateral ICV KA administration
  14. Alterations in hippocampal cytoarchitecture following unilateral ICV KA administration
  15. Mossy fiber distribution in the intact control hippocampus
  16. Aberrant mossy fiber sprouting in the adult hippocampus after unilateral ICV KA administration
  17. Discussion
  18. Relationship between neurotrophin levels and initiation of aberrant mossy fiber sprouting
  19. Association between neurotrophin levels and the period of robust mossy fiber sprouting
  20. Potential mechanisms of NT-3 and NGF involvement in aberrant mossy fiber sprouting
  21. Potential mechanisms and significance of BDNF downregulation in the chronically lesioned hippocampus
  22. Acknowledgements
  23. References
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