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Keywords:

  • α-spectrin;
  • calpain;
  • neuroprotection;
  • SNJ-1945;
  • traumatic brain injury

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The efficacy of the amphipathic ketoamide calpain inhibitor SNJ-1945 in attenuating calpain-mediated degradation of the neuronal cytoskeletal protein α-spectrin was examined in the controlled cortical impact (CCI) traumatic brain injury (TBI) model in male CF-1 mice. Using a single early (15 min after CCI-TBI) i.p. bolus administration of SNJ-1945 (6.25, 12.5, 25, or 50-mg/kg), we identified the most effective dose on α-spectrin degradation in the cortical tissue of mice at its 24 h peak after severe CCI-TBI. We then investigated the effects of a pharmacokinetically optimized regimen by examining multiple treatment paradigms that varied in dose and duration of treatment. Finally, using the most effective treatment regimen, the therapeutic window of α-spectrin degradation attenuation was assessed by delaying treatment from 15 min to 1 or 3 h post-injury. The effect of SNJ-1945 on α-spectrin degradation exhibited a U-shaped dose–response curve when treatment was initiated 15 min post-TBI. The most effective 12.5 mg/kg dose of SNJ-1945 significantly reduced α-spectrin degradation by ~60% in cortical tissue. Repeated dosing of SNJ-1945 beginning with a 12.5 mg/kg dose did not achieve a more robust effect compared with a single bolus treatment, and the required treatment initiation was less than 1 h. Although calpain has been firmly established to play a major role in post-traumatic secondary neurodegeneration, these data suggest that even brain and cell-permeable calpain inhibitors, when administered alone, do not show sufficient cytoskeletal protective efficacy or a practical therapeutic window in a mouse model of severe TBI. Such conclusions need to be verified in the human clinical situation.

Abbreviations used
CCI

controlled cortical impact

SBDP

α-spectrin breakdown product

TBI

traumatic brain injury

TBS

of tris buffered saline

The complex biochemical signaling mechanisms that make up the secondary injury response following traumatic brain injury (TBI) constitutes a major factor in determining the extent of post-traumatic neurodegeneration and neurological outcome. The compromise of calcium homeostasis is a well-established biochemical sequelae of TBI (McIntosh et al. 1997). Inducers of calcium perturbations leading to intracellular calcium overload include trauma-induced glutamate excitotoxicity and subsequent activation of glutamate receptor-operated and voltage-dependent calcium channels. The resulting accumulation in calcium induces downstream activation of enzymes and cytotoxic signaling cascades including those mediated by the cysteine protease, calpain (McCracken et al. 1999).

Activated calpains cleave key cellular substrates including cytoskeletal, membrane-associated, and neurofilament proteins and are associated with increased cell death (Kampfl et al. 1997). The neuronal cytoskeletal protein α-spectrin is a well-characterized substrate of calpain that is degraded into a calpain-specific 145-kDa α-spectrin breakdown product (SBDP), a caspase 3-specific 120 kDa SBDP or a 150 kDa mixed calpain/caspase 3 SBDP that can be differentiated and quantified by western blot (Wang 2000). Analysis of α-spectrin degradation is widely used as a measurement of post-TBI calpain activity and an assessment of biochemical neuroprotection in TBI models (Saatman et al. 1996a; Buki et al. 1999; Kupina et al. 2001, 2003; Thompson et al. 2006) and more recently as a CSF biomarker for human TBI (Farkas et al. 2005; Brophy et al. 2009).

Calpain inhibition has represented a viable neuroprotective therapeutic target for TBI because of its downstream location in the cytotoxic signaling induced by early biochemical disruptions in glutamate and Ca2+ homeostasis (Bartus 1997; Saatman et al. 2010). Thus, delayed Ca2+-induced neuropathological events such as axonal damage and neuronal death can be interjected through calpain inhibition resulting in preservation of neuronal integrity and function. Although calpain activation is an early event following TBI, it has been shown to remain active for at least 24 h days following injury (Thompson et al. 2006; Deng et al. 2007), thus theoretically allowing for a wider therapeutic post-injury time window. Furthermore, inhibition of calpain appears relatively safe as the physiological levels of active calpain are very low and the proactive form of the enzyme is only hyper-activated by calcium during pathological conditions.

There are a multitude of calpain inhibitors that have been developed and evaluated in various models of TBI including AK295 (Saatman et al. 1996b), calpain inhibitor II (Posmantur et al. 1997), SJA6017 (Kupina et al. 2001), and MDL28170 (Thompson et al. 2010). Our recent pharmacological analysis of MDL28170 in the mouse CCI-TBI model revealed the compound to be able to partially decrease calpain-mediated cytoskeletal degradation in the mouse brain after CCI-TBI injury if initially administered 15 min post-TBI followed by a pharmacokinetically appropriate maintenance dosing regimen out to 4 h. However, the anti-calpain efficacy was insufficient to produce a statistically significant effect if treatment was delayed for more than 1 h. (Thompson et al. 2010).

SNJ-1945 ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl) -3-methylbutyl) carbamic acid 5-methoxy-3-oxapentyl ester) is a more recently identified α-keto amide calpain inhibitor that is based on the backbone of SJA6017 (Kupina et al. 2001) and was designed to improve membrane permeability and stability and to increase solubility thereby increasing overall bioavailability. SNJ-1945 was originally developed as a potential orally administered therapeutic for calpain-induced retinal degeneration and dysfunction (Oka et al. 2006; Shirasaki et al. 2006, 2008), but it has recently been shown to be protective in models of focal brain ischemia and cardiac dysfunction (Koumura et al. 2008; Yoshikawa et al. 2010). In this study, we investigated the pharmacological profile of SNJ-1945 in the mouse controlled cortical impact (CCI-TBI) model. We evaluated the dose–response, optimal treatment duration, and therapeutic window of SNJ-1945 in terms of inhibition of calpain-mediated cortical neuronal α-spectrin degradation at its previously determined 24 h post-TBI peak in the mouse CCI-TBI model (Thompson et al. 2006; Deng et al. 2007).

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals

This studies employed young adult male CF-1 mice (Charles River, Portage, MI, USA) weighing 29–32 g. All animals were fed ad libitum and housed in the Division of Laboratory Animal Resources sector of the University of Kentucky Medical Center, which is fully accredited by AALAC. All procedures followed protocols approved by the University of Kentucky's Institutional Animal Care and Use Committee.

Controlled cortical impact injury

The mice were anesthetized using isoflurane (2.5%), shaved, and placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA), with the head positioned in the horizontal plane and the nose bar set at zero. Following a midline incision to expose the skull, a 4-mm craniotomy was made lateral to the sagittal suture, and centered between the lambda and the Bregma. The skull at the craniotomy site was removed without disrupting the underlying dura. A pneumatically driven piston (Precision Systems and Instrumentation; LLC, Fairfax, VA, USA) containing a 3 mm diameter flat tip that depressed the cerebral tissue 1.0 mm (3.5 m/sec velocity) for 500 ms was used to produce the CCI-TBI. After injury, the craniotomy was closed by placement of a 6.0 mm diameter disk made of dental acrylic that was cemented in place with cyanoacrylate. The mice were then placed in a Hova-Bator Incubator (model 1583; Randall Burkey Co, Boerne, TX, USA), set at 37°C, for at least 20 min to prevent post-traumatic hypothermia. All animals had ad libitum access to food and water. Further details of the procedure are available in our previous publications (Hall et al. 2008; Thompson et al. 2010).

Western blot analysis of cytoskeletal degradation

To measure calpain-specific α-spectrin degradation, the mice were euthanatized at 24 h post-impact, the time point when α-spectrin degradation is maximally elevated in both the ipsilateral cortex and hippocampus post-CCI-TBI (Thompson et al. 2006; Deng et al. 2007). The mice received an overdose of sodium pentobarbital (200 mg/kg IP). The ipsilateral cortex and hippocampus were rapidly dissected on an ice-chilled stage and immediately transferred to Triton lysis buffer (1% Triton, 20 mM Tris HCL, 150 mM NaCl, 5 mM EGTA, 10 mM EDTA, and 10% glycerol) containing protease inhibitors (Complete Mini Protease Inhibitor Cocktail; Roche Diagnostics Corp., Indianapolis, IN, USA). The samples were sonicated and then centrifuged for a period of 30 min (18 000 g at 48°C). The supernatants were collected and the pellet was discarded. Protein concentrations were determined using the BioRad DC Protein Assay, and samples were diluted to 1 mg/mL of protein. Protein samples (5 μg) were run on 3–8% Tris-Acetate Criterion™ XT Precast gels (Bio-Rad, Hercules, CA, USA), and then transferred to a nitrocellulose membrane via a semi-dry electro-transferring unit. The membranes were incubated in a blocking solution of tris buffered saline (TBS; 20 mM Tris HCL, 150 mM NaCl) plus 5% milk for 1 h. This was followed by incubation in TBST blocking solution containing mouse monoclonal anti- α-spectrin antibody (1 : 5000, Affiniti FG6090; Affiniti, Mamhead Castle, UK) or β-tubulin (1 : 10 000) overnight at 4°C. α-Spectrin positive bands were detected by a goat anti-mouse secondary antibody conjugated to an infrared dye (IRDye800CW, Rockland, Gilbertsville, PA, USA) at a dilution of 1 : 5000 and β-tubulin was detected by a goat anti-rabbit secondary antibody conjugated to an infragreen dye (1 : 10 000, IRDYE650CW; Rockland Immunochemicals, Inc.). All incubations and wash steps were performed according to the instructions given by the manufacturers. The membranes were imaged and quantified using the LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). In the current experiments, the band analyzed for α-spectrin breakdown was the calpain-specific 145-kDa band which is specific for calpain-mediated proteolysis (Wang 2000). The β-tubulin served as the loading control and was used to normalize band densities between blots.

SNJ-1945 preparation and dosing

SNJ-1945 was prepared fresh daily and suspended in distilled water containing 0.5% carboxymethyl cellulose and administered intraperitoneally (i.p.). This vehicle was chosen based on its previous use by other investigators (Koumura et al. 2008). SNJ-1945 was obtained from the Senju Pharmaceutical Co., Ltd (Kobe, Japan). For the initial dose–response experiment, mice were injected with a single i.p. dose of SNJ-1945 (6.25, 12.5, 25, or 50 mg/kg) at 15 min post-CCI-TBI. The dose range was selected based on the dose used for prior studies with i.p. administration of SNJ-1945 in a mouse model of focal cerebral ischemia (Koumura et al. 2008). The 6.25 mg/kg and 12.5 mg/kg doses were delivered using a SNJ-1945 concentration of 1 mg/ml in a volume of 0.188 mL and 0.375 mL, respectively. The 25 mg/kg and 50 mg/kg doses were delivered using a 5 mg/mL SNJ-1945 concentration in a volume of 0.150 mL and 0.300 mL, respectively. In a second experiment, we wanted to determine whether repeated administration of SNJ-1945 might produce a greater effect than the single 15 min. post-TBI i.p. dose, we gave additional doses equal to half of the initial dose at 2 h, and again at 4 h, after the initial 15 min. post-TBI dose. The dosing interval was chosen based on the 4 h plasma half-life of SNJ-1945 in rodents (Shirasaki et al. 2006).

Sample size & data analysis

All data were expressed as the mean ± SEM for n = 10/group, and analyzed using a one-way analysis of variance (anova), followed by Student-Newman-Keuls (SNK) or Bonferroni multiple comparisons post hoc analysis, as indicated. A value of p < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Dose response of SNJ-1945 on post-traumatic α-spectrin degradation

The established time course of α-spectrin breakdown is characterized by an initial increase that peaks at 6 h and remains stable out to 12 h, followed by a secondary significant increase and peak at 24 h post-CCI-TBI (Thompson et al. 2006; Deng et al. 2007). We examined the dose–response profile (6.25, 12, 25, 50 mg/kg) of early (15 min post-injury) administration of SNJ-1945 on peak post-traumatic α-spectrin degradation following CCI-TBI. Cortical tissues were harvested 24 h post-injury and subjected to α-spectrin western blot to measure SBDP levels. As expected, CCI-TBI induced a significant increase in the 145-kDa SBDP in the ipsilateral cortex (Fig. 1a, ***p < 0.001 vs. sham). The effect of SNJ-1945 on α-spectrin proteolysis followed a U-shaped dose–response curve. A one-way anova with Bonferroni multiple comparisons post hoc analysis revealed a significant 60% reduction in the 145-kDa SBDP with the 12.5 mg/kg dose compared with the vehicle group (Fig. 1a, #p < 0.05 vs. vehicle). Doubling the dose to 25 mg/kg resulted in a loss of statistical significance although the effect of that dose was nearly the same as 12.5 mg/kg. Nevertheless, the loss of significance at 25 mg/kg appears to represent the threshold for the right side of a U-shaped dose–response curve as indicated by the 50 mg/kg dose clearly showing a loss of effect compared with lower doses. Based on this data, 12.5 mg/kg was established as the most effective dose and was used as the initial treatment dose for the remainder of the study. A representative western blot of the 145-kDa SBDP from the ipsilateral cortex of animals that had been treated with SNJ-1945 is also provided in Fig. 1b.

image

Figure 1. (a) Dose–response analysis of SNJ-1945 on calpain-mediated cytoskeletal degradation in the ipsilateral cortex following controlled cortical impact-traumatic brain injury (CCI-TBI). A single i.p. injection of 6.25 mg/kg, 12.5 mg/kg, 25 mg/kg or 50 mg/kg SNJ-1945 was administered 15 min post-injury and α-spectrin degradation was analyzed via western blot at 24 h post-injury. SNJ-1945 demonstrates a U-shaped doses–response curve with 12.5 mg/kg representing the most effective dose. (b) Representative western blot demonstrates the proteolysis of α-spectrin into the 150- and 145-kDa SBDPs in sham or CCI-TBI injured mice treated with vehicle or SNJ-1945. n = 10 animals per group; values = mean ± SEM; one-way anova followed by Bonferroni multiple comparisons post hoc test: ***p < 0.001 versus sham and #p < 0.05 versus vehicle.

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In this study, SNJ-1945 was administered i.p. as a drug suspension in normal saline using 5% carboxymethylcellulose as a water soluble suspending agent as described by other investigators who reported protective efficacy of the compound in a mouse focal stroke model (Koumura et al. 2008). As noted above, a single administration dose–response analysis showed a U-shaped dose–response, which was concerning considering the drug was delivered as an emulsion. Since higher drug concentrations in suspension in an emulsion may affect the absorption rate of the drug, it was important to take into account the drug particle to volume ratio for the dose–response experiment to exclude pharmacokinetic variability because of different drug absorption rates with the varying dosages. Thus, we examined the dose–response using two methods of drug preparation; fixed drug stock concentration with a dose-related increase in the administered volume versus fixed administered drug volume with a dose-related increase in drug concentration. In Fig.  1, the SNJ-1945 stock concentration remained the same, while the volume of drug injected varied between doses.

In a separate experiment, we administered the SNJ-1945 doses using the same volume for all doses with dose-related increases in drug stock concentrations such that the lowest dose (6.25 mg/kg) contained a diluted concentration of emulsified drug particles when compared with the highest dose (50 mg/kg) which necessitated having more drug particles suspended in the same volume of vehicle. The U-shaped curve was observed using both methods of drug preparation (data not shown) demonstrating that the U-shaped dose–response effect was because of pharmacological rather than pharmaceutical (i.e., solubility) limitations.

Repeated dosing with SNJ-1945 on post-traumatic α-spectrin degradation

Effect of a three dose SNJ-1945 regimen over 4 h

Since SNJ-1945 has a half-life of about 4 h in rodents (Shirasaki et al. 2008), we determined whether a three dose regimen administered over the first four post-injury hour, which would maintain blood levels at effective calpain inhibitory levels for at least 4–6 h would decrease calpain-induced degradation of α-spectrin to a greater extent than the single dose. As previously established, CCI-TBI again induced cytoskeletal damage as measured by the significant increase in the α-spectrin specific 145-kDa SBDP in the ipsilateral cortex at 24 h post-injury (Fig. 2a, ***p < 0.001 vs. sham). Post-injury administration of SNJ-1945 in a three-dose regimen (12.5 mg/kg i.p. 15 min post-TBI and 6.25 mg/kg i.p. at 2 h 15 min and 4 h 15 min post-TBI) significantly reduced the 24 h post-injury 145-kDa SBDP in the ipsilateral cortex. However, this repeated dosing paradigm did not produce a greater reduction in α-spectrin degradation compared to administration of a single 12.5-mg/kg dose (compare Figs 1a and 2a). Furthermore, doubling the doses in the three dose regimen resulted in a complete loss of the effect (data not shown).

image

Figure 2. The effect of repeated SNJ-1945 dosing on calpain-mediated α-spectrin degradation in the ipsilateral cortex at 24 h following controlled cortical impact-traumatic brain injury (CCI-TBI). (a) In a first experiment, three-doses of SNJ-1945 were administered i.p. beginning with 12.5 mg/kg 15 min post-injury followed by two 6.25 mg/kg doses at 2 h 15 min and 4 h 15 in post-injury which significantly reduced the calpain-specific 145-kDa SBDP by ~50%. (b) In a second experiment, mice were treated with 12.5 mg/kg (i.p.) SNJ-1945 15 min post-injury followed by 6.25 mg/kg every 2 h (2 h 15 min, 4 h 15 min, 6 h 15 min, 8 h 15 min, 10 h 15 min 12 h 15 min) until 12 h post-injury. Although some attenuation of α-spectrin degradation was seen, the effect was not significant. n = 10 animals per group; values = mean ± SEM; one-way anova followed by SNK post hoc test: ***p < 0.001 and **p < 0.01 versus sham and ##p < 0.01 versus vehicle.

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Effect of an extended seven-dose SNJ-1945 regimen over 12 h

We next explored the effect of a more extended SNJ-1945 treatment regimen delivered over 12 h. The rationale was to maintain an effective calpain inhibitory plasma levels (and presumably brain levels) into the 12 to 24 h post-injury period in which there is a secondary rise in α-spectrin degradation between those time points, with 24 h being the peak of post-injury cytoskeletal degradation as we have previously shown (Thompson et al. 2006; Deng et al. 2007). Thus, a seven-dose treatment paradigm was examined (12.5 mg/kg i.p. at 15 min post-injury followed by 6.25 mg/kg i.p. at 2, 4, 6, 8, 10, 12 h after the initial dose). However, the extended SNJ-1945 treatment regimen failed to produce a significant reduction in the 145-kDA SBDP compared to vehicle (Fig. 2b, **p < 0.01 vs. sham).

Therapeutic window analysis of SNJ-1945 on calpain-mediated cytoskeletal degradation after CCI-TBI

A therapeutic window study was performed to determine whether the single 12.5 mg/kg administered dose of SNJ-1945 could maintain its effectiveness when the initial 15 min post-injury injection was delayed for a longer post-TBI period. For this experiment, we resorted back to the single i.p. dose since that approach had provided us with a better effect than either the 4 h or 12 h repeated dose regimens. Accordingly, 12.5 mg/kg of SNJ-1945 was administered i.p. beginning at 15 min, 1 or 3 h following injury. As in the previous experiments, the animals were killed at 24 h following CCI-TBI and α-spectrin degradation was analyzed via western blot. As previously observed, CCI-TBI induced a significant increase in the 145-kDa SBDP, which was reduced with 15 min post-injury administration of 12.5 mg/kg SNJ-1945 (Fig. 3, *p < 0.05 vs. sham). In contrast, SNJ-1945 was unable to maintain its early post-injury effectiveness when treatment was delayed to 1 or 3 h post-injury.

image

Figure 3. Therapeutic window analysis of SNJ-1945 as measured by calpain-mediated α-spectrin degradation 24 h post-controlled cortical impact-traumatic brain injury (CCI-TBI). Animals were treated with a single 12.5-mg/kg (i.p.) dose of SNJ-1945 at 15 min, 1 h, or 3 h post-injury. Ipsilateral cortical tissues were harvested 24 h after injury and a α-spectrin immunoblot analysis was performed. CCI-TBI significantly increased 145-kDa SBDP levels in the ipsilateral cortex at 24 h post-TBI. SNJ-1945 administered 15 min after injury reduced 145-kDa SBDP in the ipsilateral cortex. However, the effect was lost when SNJ-1945 treatment was delayed by 1 h and 3 h post-injury. n = 7 animals per group; values = mean ± SEM; one-way anova followed by SNK post hoc test: *p < 0.05 versus sham.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In this study, we assessed the efficacy of SNJ-1945-mediated calpain inhibition in the mouse CCI-TBI model by measuring the degradation of α-spectrin into the calpain-specific 145-kDa SBDP (Wang 2000) at its 24 h peak (Thompson et al. 2006; Deng et al. 2007). We first performed a dose–response analysis using a single bolus of SNJ-1945 administered 15 min post-injury to identify the most effective single dose of SNJ-1945 required to attenuate 24 h α-spectrin degradation. This experiment revealed that 12.5 mg/kg was the ideal dose. However, a U-shaped dose–response pattern was observed, wherein higher doses caused a loss of efficacy. This was followed by testing a short term 4 h and an extended 12 h multiple dosing regimen to investigate whether repeated SNJ-1945 dosing would improve on the inhibition of α-spectrin degradation. It did not, and the extended dosing regimen also resulted in a loss of an effect. Finally, we determined the therapeutic window of the single 12.5 mg/kg dose of SNJ-1945 by delaying the initial treatment time. This last experiment revealed that the cytoskeletal protective action of SNJ-1945 is less than 1 h post-injury.

A major uncertainty concerning calpain inhibition as a therapeutic strategy for TBI is derived from the reported differences among the various pharmacological inhibitors regarding inhibitory selectivity versus simultaneous inhibition of other proteases (e.g., caspase 3, cathepsins, and proteosomal), selectivity for particular calpain isoforms, brain and cellular permeability, dose–response, optimal duration of treatment and therapeutic window in established models of TBI. Conflicting data across TBI experimental studies with various calpain inhibitors have lead neuroprotection researchers to question the potential of pharmacological calpain inhibition to reliably improve experimental outcomes to a degree that makes this approach translatable to human TBI trials. Moreover, the current limitations with the available calpain inhibitors, which may contribute to the reported inconsistencies in TBI outcome measurements, are mostly pharmaceutical (solubility, stability) and/or pharmacokinetic (blood brain barrier and/or neuronal permeability). While SNJ-1945 improves on those limitations, aqueous solubility still is not ideal and more importantly, the U-shaped dose–response might be problematic in clinical studies in which the effective dose range could be narrow. This dose–response pattern has not been described for any of the calpain inhibitors previously tested in TBI models, but this is the first study to carry out a detailed dose–response analysis. Having excluded the possibility of solubility limitations as a pharmaceutical explanation for the U-shaped dose–response, we are left with the idea that there may be an unknown complexity in SNJ-1945s cytoskeletal protective pharmacology, where lower doses protect against post-traumatic calpain-mediated cytoskeletal damage, while at higher doses some opposing action causes the effect to be lost.

A series of repeated dosing experiments were performed to maintain calpain inhibition for the duration of its established peak activity in the mouse CCI-TBI model. The failure of repeated administration of SNJ-1945 every 2 h up to 12 h to reduce α-spectrin degradation is likely because of intracellular drug concentrations reaching the concentration range associated with the right side of the U-shaped dose–response curve. The observed U-shaped dose–response curve for neuronal cytoskeletal protection suggests a sensitive threshold of calpain inhibition where too much inhibition may actually contribute to counter-productive actions in injured neurons. It is plausible that calpain exhibits multimodal effects that vary depending on the needs of the surrounding at risk neuronal population in the injured brain. For instance, it has been observed that there is a requirement for calpain activity in plasma membrane wound repair following mechanical stress in embryonic fibroblasts cultured from calpain small-subunit knockout mice (Mellgren et al. 2007). Moreover, the same investigators utilized isozyme-selective siRNAs to demonstrate that calpain-mediated plasma membrane repair was indeed specific to m- or μ-calpain and not other cytosolic proteases (Mellgren et al. 2009). Therefore, in this study, we speculate that the lack of effect on cytoskeletal degradation with higher doses of SNJ-1945 could be an indirect result of the inhibition of calpain-mediated membrane reparative effects, the results of which antagonize the neuroprotective effects of inhibition of calpain-mediated cytoskeletal proteolysis.

Recently, we described experiments with the calpain inhibitor MDL28170 in the presently employed mouse CCI-TBI model in which we demonstrated neuroprotection in terms of a decrease in cortical α-spectrin proteolysis, but without a reduction in cortical contusion lesion volume (Thompson et al. 2010). Although the reduction in α-spectrin degradation with MDL28170 in that study was modest, the dose–response did not appear to follow a U-shaped curve as an extended treatment regimen of four doses over 4 h and 45 min after TBI proved more effective than administration of two doses over 45 min post-TBI. In addition, a statistically significant reduction in α-spectrin degradation remained when treatment initiation was delayed from 15 min to 1 h post-injury, but not 3 h.

It should be cautioned that this study does not include a histological evaluation of the ability of SNJ-1945 to reduce cortical contusion lesion volume or any behavioral measures related to motor or cognitive outcome. However, as noted above, our previous work with the calpain inhibitor MDL28170 showed that compound was ineffective in reducing lesion volume despite its reduction of calpain-mediated α-spectrin degradation (Thompson et al. 2010). Concerning the lack of an effect on lesion volume, it is plausible that the cytoskeletal protective effect of calpain inhibition is region and perhaps neuronal population specific. Effective inhibition of calpain-mediated α-spectrin proteolysis is likely limited to the area adjacent to the cortical contusion lesion. This area might be referred to as the ‘traumatic penumbra’ analogous to the term ‘ischemic penumbra’, which refers to the area at risk surrounding the core of the infarcted zone in focal stroke studies. Intuitively, in TBI (as well as in ischemic stroke), activation of secondary injury cascades would be delayed in this area compared to the epicenter of contusion lesion site where cell death pathways are more rapidly initiated following injury. Indeed, post-traumatic calpain activation and α-spectrin degradation have been detected as early as 15 min after TBI, which limits the possibility of pharmacologically attenuating acute calpain-mediated proteolysis at the contusion site, where secondary injury mechanisms are more intensely activated and damage evolves relatively rapidly after CCI-TBI (Kampfl et al. 1996; Saatman et al. 1996a).

Whether SNJ-1945 would decrease cortical lesion volume, the decrease in post-traumatic α-spectrin degradation clearly represents neuronal and mainly axonal cytoskeletal neuroprotection since α-spectrin is present in neuronal processes as a neurofilament-stabilizing protein. Consistent with this assertion, MDL28170, which also reduced post-traumatic α-spectrin degradation in the CCI-TBI model (Thompson et al. 2010), has also been reported in the rat fluid percussion TBI model to attenuate corpus callosal axonal degeneration as evidenced by increase axonal sparing and function (Ai et al. 2007).

Despite the axonal cytoskeletal protective effects of SNJ-1945 shown in this report, enthusiasm for the further neuroprotective evaluation of this particular calpain inhibitor appears limited due in part to the narrow U-shaped dose–response curve that may be because of negative effects of the compound on calpain-mediated membrane repair that may predominate at the upper end of the dose–response curve. Furthermore, the compound's ability to demonstrably inhibit cytoskeletal degradation is associated with a therapeutic window that is too short to be relevant to future clinical testing in TBI patients. While slightly longer, the therapeutic window for MDL28170 is also less than 3 h post-injury (Thompson et al. 2010). Although these characteristics do not completely eliminate calpain inhibition as a therapeutic approach, it would seem that their potential for TBI neuroprotection when administered alone is associated with serious limitations across the class of compounds that are direct-acting calpain inhibitors.

We have recently demonstrated in other studies in the mouse CCI-TBI paradigm that treatment with either the mitochondrial neuroprotectant NIM811 (Mbye et al. 2009) or the lipid peroxidation inhibitor U-83836E (Mustafa et al. 2011), which both act to protect mitochondrial calcium buffering, can indirectly limit post-traumatic calpain activation and attenuate α-spectrin degradation with as much as a 12 h post-traumatic therapeutic window. Thus, the approach of protecting cellular homeostatic mechanisms that indirectly limit calpain activation by controlling intracellular calcium levels may be a promising approach for inhibiting the neurodegenerative effects of calcium overload-activated calpain activation in TBI than trying to inhibit calpain directly beyond the first few hours after injury. Nevertheless, it is possible that the addition of a direct calpain inhibitor such as SNJ-1945 might further improve the protective effects of mitochondria protectants or antioxidant compounds, as a combinatorial neuroprotective approach. This remains to be determined in future studies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study was supported by funding from the National Institute of Neurological Disorders and Stroke (2P01 NS058484, 2P30 NS051220) and the Kentucky Spinal Cord & Head Injury Research Trust. The authors have no conflicts of interest to declare in relation to the contents of this manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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