Effects of Reg-2 on Survival of Spinal Cord Neurons In Vitro
Article first published online: 20 JAN 2010
Copyright © 2010 Wiley-Liss, Inc.
The Anatomical Record
Volume 293, Issue 3, pages 464–476, March 2010
How to Cite
Fang, M., Huang, J.-Y., Ling, S.-C., Rudd, J. A., Yew, D. T. and Han, S. (2010), Effects of Reg-2 on Survival of Spinal Cord Neurons In Vitro. Anat Rec, 293: 464–476. doi: 10.1002/ar.21087
- Issue published online: 17 FEB 2010
- Article first published online: 20 JAN 2010
- Manuscript Accepted: 26 OCT 2009
- Manuscript Received: 9 DEC 2008
- National Natural Science Foundation of China. Grant Number: 30600192
- cell culture;
- lactate dehydrogenases;
- hydrogen peroxide;
- azide sodium;
- mitochondrial poisoning
Regeneration gene protein 2 (Reg-2) is a small secreted protein expressed in motor and sensory neurons of spinal cord during developmental stages and following injury of peripheral nerves. Reg-2 appears to act as a neurotrophic factor and protects injured neurons from death during regeneration. To illustrate these potential protective effects in vitro, we investigated the blocking effects of Reg-2 antibodies on the survival of primary cultured spinal cord neurons and astrocytes, as well as on neurite outgrowth. In addition, the effects of Reg-2 in neuron injury models induced by peroxide and mitochondrial poisoning were assessed. Our results showed that Reg-2 antibody markedly reduced survival and neurite outgrowth from neurons, whereas astrocyte survival was unaffected. Addition of Reg-2 into the culture medium had no effect on neuron survival or neurite outgrowth. However, the addition of the Reg-2 into culture media after peroxide treatment or cellular hypoxia insult induced by mitochondrial poisoning can reduce lactate dehydrogenase release levels and cell death. Thus, the data suggests that Reg-2 is essential for the survival and neurite outgrowth of developing spinal cord neurons but not the survival of glial cells, and that Reg-2 plays protective effects on spinal cord neurons against injury in vitro. Anat Rec, 2010. © 2010 Wiley-Liss, Inc.
Reg-2/PAP I is a member of the pancreatitis-associated protein (PAP) family (Okamoto, 1999). It is a small (16 kDa) secretory protein induced during acute pancreatitis but not normally expressed in the healthy tissue (Iovanna et al., 1991). PAP was considered to play a role as an hepatic cytokine involved in hepatic proliferation and survival and also in liver regeneration (Iovanna et al., 1991; Orelle et al., 1992; Ortiz et al., 1998; Malka et al., 2000; Lieu et al., 2007). PAP also has antibacterial and antiapoptotic properties, which can against pancreatitis and liver injury (Terazono et al., 1988; Simon et al., 2003). Previous studies also revealed the potential role of PAP in the development and regeneration of the central and peripheral nervous system (Livesey et al., 1997; Nishimune et al., 2000).
There are reports that Reg-2 is expressed in mice in a cytokine-dependent manner in both sensory and motor neurons during embryo development (Livesey et al., 1997). The expression is transient and declines in postnatal developmental stages and is consequently absent in normal adults. In rats, Reg-2 is also constitutively expressed in subpopulations of motor neurons during development (Nishimune et al., 2000). This expression is driven by cytokines of the interleukin-6 family, including ciliary neurotrophic factor (CNTF), leukemia inhibitory factor, and cardiotrophin (Dusetti et al., 1995; Livesey et al., 1997; Averill et al., 2002; Schweizer et al., 2002). Finally, Reg-2 also acts as a neurotrophic factor that is an essential intermediary via the CNTF pathway to support the survival of motor neurons during development (Nishimune et al., 2000).
Although Reg-2 is not normally expressed in the adult central or peripheral nervous system, it is rapidly upregulated following crushing or transection of peripheral motor and sensory nerves, (Averill et al., 2002; Namikawa et al., 2005). Neutralizing Reg-2 activity in vivo by intraneural injection of anti-Reg-2 polyclonal antiserum at the site of peripheral nerve damage leads to a reduced level of axonal regeneration, which suggests that Reg-2 may have an important role in regenerative processes (Livesey et al., 1997).
In Reg-2 knockout mice, the deletion of the RegIII gene, which is equal to Reg-2 in rats, did not affect the motor neuron survival studied up to 28 weeks after birth, although there was no CNTF-mediated rescue of neonatal facial motor neurons after axotomy when compared with wild-type mice. However, the dynamics of Reg-2 expression in rats are different from that of RegIII in mice. Thus, RegIII does not reappear in the adult mouse after transection of the facial or sciatic nerve, and it does not decrease 24 hr after axotomy, as seen following sciatic transection in neonatal rats (Averill et al., 2002; Schweizer et al., 2002; Tebar et al., 2008).
We previously showed that Reg-2 expression increases in motor and sensory neurons in an adult rat spinal cord transection model. The expression was undetectable in oligodendrocytes and astrocytes but peaked 3 days after injury in neurons of dorsal horn and 7 days in neurons of ventral horn (Xu et al., 2008). In this investigations, we aim to determine if the survival of primary cultured neurons and astrocytes from embryonic spinal cord could be affected by neutralizing Reg-2 activity with Reg-2 antibodies in vitro, and if the addition of Reg-2 proteins would further enhance the survival and neurite outgrowth of cultured neurons. The studies were extended to also investigate the potential neuroprotective action of Reg-2 on cultured neurons after peroxide insult and cellular hypoxia induced by mitochondrial poisoning.
MATERIALS AND METHODS
Immunofluorescent Staining and Double Immunofluorescent Staining
Embryonic S-D rats at 18 days of gestation (E18) were removed from their pregnant mothers, and the whole intact spinal cords of embryos were taken out, fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PH 7.4). Twenty-μm-thick sections were cut on freezing microtome through horizontal plane and transverse plane. Sections were collected in 0.01 M PBS and mounted on 0.02% poly-L-lysine-coated slides.
Adjacent sections were processed for immunofluorescent staining for Reg-2 or double-immunofluorescent staining for Reg-2 and NGFR, a specific marker of developing motor neuron. Slides were warmed for 20 min on a slide warmer and rinsed in 0.1 M Tris-buffered saline (TBS) for 10 min. After blocking nonspecific staining with 10% goat serum in TBS containing 0.3% Triton X-100 (TBST) for 1 hr at room temperature, sections were incubated overnight at 4°C in TBST containing 5% goat serum and mouse anti-Reg-2 (1:50, R&D system, MN), rinsed in 0.1 M TBS, incubated in TBST containing 5% goat serum and 1:200 TRITC (Rhodamine)-conjugated goat antimouse IgG secondary antibodies (Invitrogen, Carlsbad, CA). Then, double-stained with rabbit anti-NGFR (1:200 Thermo Fisher Scientific, CA) at 4°C overnight, incubated in 1:200 of FITC (Fluorescein)-conjugated goat antirabbit IgG secondary antibody (Invitrogen, Carlsbad, CA) for 1 hr at room temperature. Finally, the sections were coverslipped with antifade Gel/Mount aqueous mounting media (Southern Biotech, Birmingham, AL). In between steps, sections were washed three times 10 min in TBS.
Briefly, total proteins were extracted from the intact spinal cord of E18 rats, the intact and injuried cultured neurons with 2 mM PMSF in 1 mL ice-cold RIPA buffer added protease inhibitor cocktail EDTA-free. The protein content was determined by the method of Bradford using the UV-2401 PC spectrophotometer (Shimadzu Co., Japan). After heating at 95°C for 5 min, SDS-PAGE were performed on 15% polyacrylamide slab gel and separated proteins were then electrophoretically transferred to PVDF membrane at 70 V for 1.5 hr at 4°C in a Bio-Rad TransBlot apparatus. After blocking nonspecific binding sites with bovine serum albumin, each membrane was incubated for 12 hr at room temperature with purified Reg-2 monoclonal (2 μg/ml) and washed five times with 0.05% Tween 20 in PBS. The membranes were then incubated at 37°C for 1 hr with HRP-conjugated goat antimouse secondary antibody (1:5000, Santa Cruz, CA), washed in TBST, and peroxidase-based detection was performed with enhanced chemiluminescence (ECL) detection reagent (Amersham Biosciences, Piscataway, NJ) followed by imaging and quantification of protein bands using Bio-Rad Quantity One 1-D software. To normalize protein bands to a gel loading control, membranes were washed in TBST and reprobed with rabbit anti-β-actin (1:5,000, AbCam, MA) followed by incubation with peroxidase-conjugated goat antirabbit (1:5,000, Santa Cruz, CA) and ECL detection.
Quantitative Real-time RT-PCR Analysis
RNA was extracted from intact spinal cord of E18 rats with Trizol (Invitrogen). Quantitative real-time PCR was performed with SYBR premix Ex Taq kit (TaKaRa, Tokyo, Japan) and ABI 7500 Real-time PCR System (Applied Biosystems); GAPDH was used as internal control. Primer sequences are as follows:
Reg (ACCESSION S43715) gene: 5′-GAGCAGAAAGATGATGAGAGT-3 (forward), 5′-TGAAACAGGGCATAGCAGT-3′ (reverse), the sizes of resulted amplicons were 192 bp. GAPDH gene: 5′-GACAACTT TGGCATCGTGGA-3′ (forward), 5′-ATGCAGGGATGATGTTCTGG-3′ (reverse), the sizes of resulted amplicons were 133 bp. All PCR consisted of a first denaturizing cycle at 94°C for 5 min, followed by an amplification profile of 40 reaction cycles with a first denaturizing cycle at 94°C for 15 sec, followed by 60°C for 45 sec, and finally detected at 60°C for fluorescent to make quantitation curve and dissociation curve.
Primary Cell Culture
Spinal cord was isolated from SD rat embryos at embryonic day 18 (E18). The tissue was then rinsed in Hank's buffered saline solution, cut into small pieces, digested with trypsin, dissociated with a fire-polished glass pipette and centrifuged to separate undissociated tissue. Cells were resuspended and plated onto poly-D-lysine-coated 6 mm glass cover slides placed within 24 wells cell culture plates. A total of 25,000 cells were then seeded onto individual cover slides in serum-free neurobasal medium supplemented with 2% B27 and 0.05 mM glutamine to observe survival and neurite outgrowth. This culture contained both neurons and glial cells. No antimitotic agents were added into the medium, but the serum-free neurobasal medium was used to selectively increase neuronal population. The presence of glial cells in cultures was allowed to provide an optimal milieu for growing neurons in culture.
Neural Survival and Neurite Outgrowth Assays In Vitro
On the 2nd day, one of the following proteins or antibodies was added into each culture: 1) Reg-2 protein (100 μg/L, Genway, CA), 2) Reg-2 antibody (10 mg/L, R&D system, MN), 3) heat-inactivated Reg-2 antibody (10 mg/L, heated at 100°C for 10 min), or 4) no protein as the antibody control. On Day 7, cultures were fixed in 4% paraformaldehyde and processed for Neurofilament M (NF-M, United States Biological, MA) and glial fibillary acidic protein (GFAP, Thermo Fisher Scientific, CA) immunofluorescent staining to identify neurons and astrocytes, respectively. The nuclei of all cells were stained with a Hoechst 33342 nuclear dye. Briefly, to identify the astrocytes, the cultures of each group were rinsed in PBS and incubated with the GFAP primary antibody (1:200) containing 1% bovine serum albumin (BSA) in TBST overnight at 4°C. On the 2nd day, cultures were washed in PBS, incubated in TBST containing 5% goat serum and 1:200 of FITC-conjugated goat antirabbit IgG secondary antibody (Invitrogen, CA,USA) for 1 hr at 37°C. To identify neurons, cultures of each group were incubated with the 160 kDa NF-M primary antibody (1:1000) overnight at 4°C, then washed with PBS and incubated with 1:200 TRITC-conjugated goat antirabbit IgG secondary antibodies for 1 hr at 37°C (Invitrogen, CA). The cultures were finally coverslipped with antifade Gel/Mount aqueous mounting media (SouthernBiotech, AL) supplemented with Hoechst 33342 nuclear dye (1 μg/μl, Sigma, MO). All control cultures were incubated in PBS without primary antibodies.
To identify the expression of Reg-2 and its colocalization with GFAP and NF-M, the culture of the control group was incubated with mouse anti-Reg-2 antibody (1:50) overnight at 4°C, and then incubated with TRITC- or FITC-conjugated goat antimouse IgG secondary antibodies (Invitrogen, CA), respectively, for further double staining with GFAP or NF-M primary antibodies.
For counting the number of NF-M-positive neurons and GFAP-positive astrocytes, eight visual fields from each coverslip was randomly selected under 400× magnification. The percentage of NF-M-positive neurons or GFAP-positive astrocytes was calculated according to the following formula using the total counted number of blank control group as 100%:
The percentage of surviving neurons = the number of NF-M-positive cells in each group/the number of NF-M-positive cells in the blank control group × 100.
The percentage of surviving astrocytes = the number of GFAP-positive cells in each group/the number of GFAP-positive cells in the blank control group × 100.
To calculate neurite outgrowth from neurons, an unbiased 10 mm counting frame containing 20 × 20 grids was superimposed on images of neurons and neurites under the microscope. Eight neurons from each culture were randomly selected under 400× magnification, and the number of intersections of each neurite were counted using grid lines. The average neurite length per neuron was analyzed using the following formula: L = π/2 × d × J in which L is the neurite length in μm, d is the vertical distance between two grid lines, and J is the number of intersections between grid lines and neuritis (Novitskaya et al., 2000).
LDH Release From Cultures After Peroxide and Hypoxia Insults
E18 spinal cords were isolated from SD rat embryos. The tissue digestion and cell dissociation procedures were the same as described earlier. Dissociated cells were seeded onto 96-well cell culture plates at a density of 1 × 106 cells/mL and grew in serum-free neurobasal medium supplemented with 2% B27 and 0.05 mM glutamine. On the 5th day, H2O2 (25 μM H2O2/L medium) and sodium azide (3 mM sodium azide/L medium) were added to the medium to induce peroxide and mitochondrial poisoning insults, respectively. In the injury groups, only the insulting factors were added to the culture medium, whereas in the treatment groups, Reg-2 protein (500 mg/L) was added into the culture medium at the same time when the insulting factors were added. In addition, the addition of 0.1% TBST into culture was used as a positive control, and a blank control (the addition of neither insulting factors nor Reg-2 protein) was also used. Each group had six duplicate wells. The cultures were maintained for three additional days after various treatments before the culture medium of each well was removed for LDH release assay using a LDH-cytotoxicity assay kit (Biovision Inc, Palo Alto, CA) according to the manufacturer's protocol. The relative absorbance of all samples was measured at 490 nm with an ELX 800UV microtiter plate reader (Bio-Tek Instruments, Winooski, VT). The measurement was repeated three times at 5-sec intervals, and the numbers of each group were calculated with the following formula:
Cytotoxicity (%) = (test sample − blank control)/(positive control − blank control) × 100
The cells left in culture wells were used for the following experiments:
- 10.4% trypan blue staining to quantify cell death in each group.
- 2NF-M-immunofluorescence labeling to quantify the number of surviving neurons in each group using the same method described earlier.
- 3Extracting total protein from both H2O2 and sodium azide insulting groups to perform western blot analysis, the procedures were the same as described above. The total protein of intact cultured neurons was taken as normal control, β-actin was used as internal control.
The percentage of surviving neurons/astrocytes, the lengths of all neurites in each neuron, and LDH release were expressed as mean ± SD. A one-way ANOVA was used to compare different groups, with post-hoc t-tests. A level of P < 0.05 was considered to be statistically significant.
The expression of Reg-2 on intact spinal cord during late stage of development can be detected in the intact spinal cord taken from E18 rats both from protein and mRNA level (Figs. 1–3). Reg-2 expression was shown in the neural cells from both ventral and dorsal horn (Fig. 1E,F), and the expression of Reg-2 was not limited in NGF-R immunolabeled motor neurons subpopulation (Fig. 1). The primary cultures of neurons taken from E18 spinal cords expressed Reg-2 in cytoplasm and within their neurites (Fig. 4A–D). However, there was no positive labeling inside the cultured astrocytes (Fig. 4E,F). No apparent morphological differences were found between any cells treated with the Reg-2 protein and control groups (Figs. 5, 6). The percentage of surviving neurons and average length of their neurites of the Reg-2 treatment group were also close to those of the blank control group (P > 0.05)(Fig. 7A,C).
In contrast, when Reg-2 antibodies were added to the culture medium, immunofluorescence detection indicated that the number of neurons had markedly declined, whereas the number of astrocyte was not affected (P < 0.01) (Fig. 6). At the light microscopic level, numerous fragmentations and degenerated cells with condensed cell bodies and shrinked neurites were observed (Fig. 5C). The number of surviving neurons after Reg-2 antibody-treatment was only 30.4% of the blank control (P < 0.01), but the numbers of surviving astrocytes was 117.5% of the respective blank control group (P > 0.05) (Fig. 7A,B). Further, the average length of neurites in Reg-2 antibody-added group was reduced when compared with the control group (P < 0.01) (Fig. 7C). Such a reduction was prevented when the antibodies were inactivated by heat before being added to culture (P > 0.05) (Fig. 7A,C). Moreover, the survival of astrocytes in the inactive antibody group did not show significant differences (P > 0.05; when compared with the control and protein-added groups) (Fig. 7B). There was also no distinct morphological difference under both light and immunofluorescent microscopy, which suggested that the inactived antibody had no obvious detrimental effect on astrocytes and on survival and neurite outgrowth of cultured neurons (Figs. 5, 6).
After either a peroxide (25 μM H2O2/L medium) or mitochondrial poisoning (3 mM sodium azide/L medium) insult, the average value of LDH cytotoxicity in these groups was close to that of the positive control group treated with 0.1% TBST (P > 0.05) (Fig. 8A). When Reg-2 protein was added to the cultures immediately after the insults, the average value of cell cytotoxicity declined significantly when compared with the average cytotoxicity level of either no Reg-2-treated or the positive control groups (P < 0.01) (Fig. 8A). When H2O2 and sodium azide insults were applied to the culture, a substantial cell death of neurons was found by both the trypan blue and immunofluorescent assay. These two insult groups had numerous trypan blue-stained dead cells (Fig. 9). The peroxide injured neurons lost their normal appearance and neurites and appeared to have bulged, swollen cell bodies and bloated nuclear bodies. The sodium azide insulted cells showed blurred, broken cell bodies with shortened neurites (Fig. 10). In contrast, the Reg-2 treatment groups successfully reduced the insult-induced neuronal death, and the surviving cells showed relatively normal appearance (Figs. 8B, 9, 10).
The results of western blot showed the expression level of Reg-2 protein remarkably decreased after the cultured neurons were insulted by mitochondrial poisoning and peroxide factors, as compared with the intact cultured neurons (Fig. 11).
The expression of Reg-2 in intact spinal cord tissue of E18 rats has been detected in our previous studies, which is consistent with earlier reports of Reg-2 using whole-mount in situ hybridization techniques in E18 rats (Nishimune et al., 2000). Reg-2 expression can be detected in the neural cells from both ventral and dorsal horn. The colocalization of Reg-2 with the NGFR-positive cells (a specific marker for developing motor neuron) is also consistent with the report of Nishimune et al. (2000). The appearance of Reg-2 positive neurons in dorsal horn, which did not show NGFR signal, suggest that the expression of Reg-2 is not limited to subpopulations of motor neurons during development. Real-time quantitative RT-PCR and western blot also confirmed the expression of Reg-2 could be detected in intact spinal cord both in protein and mRNA level during late stage of development.
A previous investigation using cultured E14 motoneurons had shown that Reg-2 expression could be induced by neurotrophic factors (Nishimune et al., 2000). By studying the present studies, we demonstrated that Reg-2 labeling in E18 neurons can also be observed without the addition of extrinsic factors. One possible reason is that neurons taken from later developmental stage (E18) are more mature, and more independent on target-derived neurotrophic factors, including some specific peptide synthesize. In addition, the model we used was a mixed culture of motor and sensory neurons, although the significance of this needs to be explored further.
The importance of this studies was to reveal that the addition of extrinsic Reg-2 provided no further increase of neuronal survival or neurite outgrowth. This may indicate that Reg-2 has no effect on cells prepared from E18 spinal cords because programmed death of neurons has already occurred (Oppenheim, 1991), and/or the autocrine/paracrine endogenous Reg-2 of the surviving neurons was already maximal for further development at this stage. However, after blocking the endogenous Reg-2 with specific antibodies, we found a significant reduction of neuronal survival and neurite outgrowth. These results confirmed the neurotrophic effects of Reg-2 and showed it is still essential for the survival and neurite outgrowth of neurons at the late stage of embryonic development.
Our study showed that E18 spinal cord neurons can express Reg-2 both in cytoplasm and in neurites, whereas no obvious Reg-2 expression was detected in astrocytes, which is consistent with pervious studies (Livesey et al., 1997; Nishimune et al., 2000). Reg-2 was not expressed in developing astrocytes and specific Reg-2 antibodies had no disastrous effects on the survival of astrocytes, suggesting the development of glial cells is relatively independent of the known neurotrophic effects of Reg-2. The observation that heat-inactivated Reg-2 antibodies had no blocking effect further confirms the neuroprotective function of the Reg-2.
Cytotoxic cell death is classically evaluated by quantification of plasma membrane damage (Mukhin et al., 1998). A standard method to measure the degree of cytotoxicity is to measure the level of LDH released from damaged cells (Regan and Guo, 1999). In previous studies, we established the mature peroxide—and mitochondrial poisoninginduced cellular hypoxia—injury models in vitro in which the injuries were induced by 25 μmol/L H2O2 and 3 mmol/L sodium azide, respectively (Han et al., 2004). Reg-2 decreased the LDH release in both models indicating its neuroprotective potential.
To illustrate the change of the Reg-2 expression following the challenges, Western blot analyses for Reg-2 was performed on the cultured spinal cord neurons with or without the azide and peroxide insults. The results showed these insults can induce remarkable decline in Reg-2 expression and have the same effects as neutralization of endogenous Reg-2. This phenomenon suggests that when the doses of insult factors reach a potential threshold, their devastating effects on mitochondria, endoplasmic reticulum, ribosome, Golgi apparatus, and other cellular organs may also have destroyed the functions of the self-production and autocrine/paracrine of some important neurotrophic factors. Thus, the expression and secretion of endogenous Reg-2 protein will decrease in injured neurons, and the supply of exogenous Reg-2 will be helpful following challenge.
Trypan blue staining and NF-M immunofluorescence labeling further confirmed the detrimental effects of the peroxide and hypoxia insults on neuronal survival and the rescuing effects of Reg-2 protein, which is also consistent with the phenomenon that blocking endogenous Reg-2 induces cell death.
In some pathological conditions and degenerative disease such as trauma and Alzheimer disease, Reg-2 expression appears unregulated (Ozturk et al., 1989; de la Monte et al., 1990; Duplan et al., 2001; Averill et al., 2008). Results of previous studies indicated that Reg-2 expression after injury may play an important role in the regenerative process, and it may represent a clinically important target in neurodegenerative disease. The Reg protein family has multiple properties, including direct axonal regenerative properties, stimulating of cell survival pathway, which reduces axonal and/or neuronal loss, a mitogenic action on Schwann cells, and anti-inflammatory properties. The Reg proteins can pass through the blood brain barrier and upregulate other members of the Reg gene family (Livesey et al., 1997; Livesey and Hunt, 1998; Hartupee et al., 2001; Averill et al., 2002; Namikawa et al., 2005). Any one or more of these properties may lead to the beneficial effects seen in animal models of CNS injury and disease.
Although this study demonstrated that Reg-2 has beneficial effects on the survival of developing neurons in vitro and protective effects against mitochondrial poisoning- and peroxide-induced injuries, its effects on the protection and functional recovery in injured animal models in vivo are unclear. In further studies, we will analyze whether Reg-2 has a neuroprotective effect in vivo using a spinal cord transection model, so as to evaluate the possibility of developing new repair strategies for the treatment of spinal cord and other CNS injuries.
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