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. 2008 Oct 21:1236:206-15.
doi: 10.1016/j.brainres.2008.07.124. Epub 2008 Aug 12.

Prevention of axonal injury using calpain inhibitor in chronic progressive experimental autoimmune encephalomyelitis

Getaw Worku Hassen  1 , Jason Feliberti, Leo Kesner, Alfred Stracher, Foroozan Mokhtarian
Affiliations

Prevention of axonal injury using calpain inhibitor in chronic progressive experimental autoimmune encephalomyelitis

Getaw Worku Hassen et al. Brain Res. .

Abstract

Axonal injury is the major correlate of permanent disability in neurodegenerative diseases such as multiple sclerosis (MS), especially in secondary-progressive MS which follows relapsing-remitting disease course. Proteolytic enzyme, calpain, is a potential candidate for causing axonal injury. Most current treatment options only target the inflammatory component of MS. Previous work using calpain inhibitor CYLA in our laboratory showed significant reduction in clinical sign, demyelination and tissue calpain content in acute experimental autoimmune encephalomyelitis (EAE). Here we evaluated markers of axonal injury (amyloid precursor protein, Na(v)1.6 channels), neuronal calpain content and the effect of CYLA on axonal protection using histological methods in chronic EAE [myelin oligodendrocyte glycoprotein (MOG)-induced disease model of MS]. Intraperitoneal application of CYLA (2 mg/mouse/day) significantly reduced the clinical signs, tissue calpain content, demyelination and inflammatory infiltration of EAE. Similarly, markers for axonal injury were barely detectable in the treated mice. Thus, this novel drug, which markedly suppresses the disease course, axonal injury and its progression, is a candidate for the treatment of a neurodegenerative disease such as multiple sclerosis.

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Figures

Figure 1
Figure 1
Changes in daily ACS of all groups over time during the course of treatment with 2mg CYLA following induction of EAE. EAE was induced in C57/Bl6 mice with MOG/CFA emulsion. CYLA, a total of 2mg/mouse/day (1mg per injection was applied i.p. twice daily). Early neurological signs such as floppy tail and hind limb weakness appeared in general about day 12pi. Over the course of time mice exhibited hind limb paralysis and some weakness in the fore limbs between days 14 and16pi. Their neurological deficit increased progressively in all EAE mice and reached its peak by day 20pi. After day 20pi until day 30pi EAE mice more or less maintained their ACS. Treatment with a total of 2mg CYLA per mouse per day starting from day 12pi led to suppression of the clinical signs to a significant level. Treatment with a total of 2mg CYLA per mouse per day starting from day 7pi caused a delayed onset and significant improvement of the clinical signs, slowing disease progression. No control groups (naïve, CFA and CYLA) exhibited neurological deficits. The experiment was terminated on day 31pi. Each graph presents daily ACS ± S.E.M. The difference between treatments and naïve control was considered significant at *P<0.05.
Figure 2
Figure 2
Qualitative assessment of axonal integrity of the lumbar spinal cord using silver impregnation method for all groups. EAE was induced in C57Bl/6J mice with MOG/CFA emulsion. CYLA, a total of 2mg/mouse/day (1mg per injection was applied i.p. twice daily). Silver staining was performed on 6–10μm cryosections. The spinal cord sections of all control groups [naïve (n=4), CFA (n=6) and CYLA (n=4)] showed normal axonal morphology (A). Extensive axonal damage (arrows) was highlighted in the spinal cords of chronic EAE mice (B). Preserved axonal morphology was observed after CYLA treatment (C). 20X magnification. Representative section from EAE mice with signs of axonal injury such as axonal bulbs, spheroids and ovoids (arrow heads, D, 40X magnification). The staining pattern among all control groups was similar. No difference was observed between the treatment groups. Nine consecutive slides from each group were assessed and every third slide from each group was chosen randomly for analysis. The mean of the selected slides from the individual groups were compared with the means of the other groups. The final result was represented as a bar graph (E). The difference between treatments and control was considered significant at *P<0.05.
Figure 3
Figure 3
Evaluation of axonal injury using APP in control, EAE and CYLA-treated groups. EAE was induced in C57Bl/6J mice with MOG/CFA emulsion. CYLA, a total of 2mg/mouse/day (1mg per injection was applied i.p. twice daily). IF labeling was performed on 50μm cryosections. Disruption of axonal transport leads to accumulation of APP within the injured axons. The spinal cord sections of all control groups [naïve (n=4), CFA (n=6) and CYLA (n=4)] showed no APP labeling (A). Extensive accumulation of APP was visible in spinal cord of chronic EAE (B). APP immunoreactivity in sections of CYLA-treated mice (day 7pi group) was similar to the control group (C). Similar result as in day 7pi groups was observed in day 12pi groups. The quantitative data is graphically represented through pixel analysis using image analyzing software ImageJ from NIH. Pixels were quantified as total number of pixels above background. Nine consecutive slides from each group were assessed and every third slide from each group was chosen randomly for pixel analysis. The mean of the selected slides from the individual groups were compared with the means of the other groups. The final result was represented as a bar graph. The difference between treatments and control was considered significant at *P<0.05 (D). 120X magnification with oil.
Figure 4
Figure 4
Differences in Nav1.6 expression in the axons of lumbar spinal cord from control, EAE and CYLA-treated groups. EAE was induced in C57Bl/6J mice with MOG/CFA emulsion. CYLA, a total of 2mg/mouse/day (1mg per injection was applied i.p. twice daily). IF labeling was performed on 50μm cryosections. The spinal cord sections of all control groups [naïve (n=4), CFA (n=6) and CYLA (n=4)] showed normal scattered distribution of Nav1.6 channels corresponding to the areas of the node of Ranvier (A). Increased and clustered Nav1.6 channels in areas of demyelination in EAE mice spinal cord were observed. Similar expression of Nav1.6 channels in spinal cords of treated mice as in control group (D). The results from day 7pi and day 12pi groups were similar. The quantitative data is graphically represented through pixel analysis using image analyzing software ImageJ from NIH. Pixels were quantified as total number of pixels above background. Nine consecutive slides from each group were assessed and every third slide from each group was chosen randomly for pixel analysis. The mean of the selected slides from the individual groups were compared with the means of the other groups. The difference between treatments and control was considered significant at *P<0.05 (D). 120X magnification with oil.
Figure 5
Figure 5
Fluorescent double-labeling using anti-calpain and anti-NeuN antibodies to identify the distribution of calpain within the neurons in chronic EAE. EAE was induced in C57Bl/6J mice with MOG/CFA emulsion. CYLA, a total of 2mg/mouse/day (1mg per injection was applied i.p. twice daily). IF with NeuN (neuronal marker) and calpain was performed on 50μm cryosections. Normal calpain distribution was visible as red dots within NeuN-positive neuron of all control groups [naïve (n=4), CFA (n=6) and CYLA (n=4)] (A). Increased calpain expression was observed within NeuN-positive neurons of EAE mice (B). The calpain distribution within NeuN-positive spinal cord neurons of CYLA-treated mice (day 7pi) was similar to control groups (C). The results of day 7pi and day 12pi groups were similar. The quantitative data is graphically represented through pixel analysis using image analyzing software ImageJ from NIH. Pixels were quantified as total number of pixels above background. Nine consecutive slides from each group were assessed and every third slide from each group was chosen randomly for pixel analysis. The mean of the selected slides from the individual groups were compared with the means of the other groups. The difference between treatments and control was considered significant at *P<0.05 (D). 120X magnification with oil.
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