Unlabeled Lumbar Spinal Cord Tissue Biology Essay

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Unlabeled lumbar spinal cord tissue omission of both primary and secondary antibodies demonstrated a negligible autofluorescence of the tissue. Primary antibody (anti-gephyrin, anti-calbindin D28K, anti-SOD1) omission control demonstrated a minimal immunoreactivity of the utilized secondary antibody (CY3- conjugated donkey anti rabbit, FITC-conjugated donkey anti-mouse), immunolabeling result was nearly comparable to unlabeled tissue (Figures 1C, 1D).

Mouse spleen tissue was utilized as a positive control for immunolabeling with cleaved caspase-3 (rabbit polyclonal, 1:100, Abcam Company) by immunoperoxidase technique. Mouse spleen demonstrated strong cytoplasmic cleaved caspase-3 labeling of spleenocytes (Figure 15A), while subsequent omission of the primary antibody (cleaved caspase-3) resulted in complete absence of spleenocytes labeling (Figure 15B).

Genotyping control

Primers control methodology was implemented to Asses the specificity of the primers utilized in the genotyping protocol. Genotyping procedures implemented on tail tissue of a confirmed non-transgenic animal demonstrated no detectable G93A-SOD1 transgene amplification (Figure 2H). Furthermore, deletion of the primers from the genotyping protocol resulted in complete absence of G93A-SOD1 transgene amplification in a confirmed positive animal (Figure 2G). Genotyping of a confirmed G93A-SOD1 transgenic animal demonstrated the amplification of the G93A-SOD1 transgene in region of 236 bp (Figure 2I).

The transgenic mouse model (G93A-SOD1), used in this study, exhibited first sign of motor neuron disease (hyperreflexia and tremor of hind limbs) around 90 days of age with hind limbs paresis around 110 days of age, proceeded to progressive paralysis which rendered animals quadriplegic around the age of 150 days.

Renshaw interneurons count.

Renshaw interneurons were enumerated in the ventral horns of every third transverse section obtained from lumbar spinal cord. This sampling method provided unbiased estimate of the Renshaw interneurons number within the lumbar spinal cord, with lower double-counting error.

Renshaw interneuron identified by its characteristic gephyrin labeling (intense, very large, and abundant gephyrin-Immunoreactive clusters on their soma and proximal dendrites) and strong calbindin D28K immunolabeling (Figure 3). Cell profiles demonstrated the above-mentioned criteria were putatively identified as Renshaw interneurons.

At 28 day of age, Renshaw interneurons mean count in transgenic animals (Figure 4B) was 2.98 per ventral horn (n=4, S.D=1.51, 294 ventral horn analyzed), compared to age-matched control animals (Figure 4A) mean count of 2.94 per ventral horn (n=4, S.D=1.62, 320 ventral horn analyzed). Statistical analysis (Figure 4C) illustrated no significant reduction in numbers of Renshaw interneurons in transgenic animals compared to age-matched control animals (Mann-Whitney Rank Sum test, p=0.79, power of test 0.8).

At 40 day of age, Renshaw interneurons mean count in transgenic animals (Figure 5B) was 1.88 per ventral horn (n=6, S.D=1.51, 399 ventral horns analyzed), compared to age-matched control animals (Figure 5A) mean count of 2.97 per ventral horn (n=6, S.D=1.62, 610 ventral horns analyzed). Statistical analysis (Figure 5C) demonstrated a significant reduction in numbers of Renshaw interneurons in transgenic animals compared to age-matched control animals (p=0.001, Mann-Whitney Rank Sum test).

At 70 day of age, Renshaw interneurons mean count in transgenic animals (Figure 7B) was 1.96 per ventral horn (n=6, S.D=1.44, 680 ventral horns analyzed), compared to age-matched control animals (Figure 7A) mean count of 3.29 per ventral horn (n=6, S.D=1.79, 618 ventral horns analyzed). Statistical analysis (Figure 7C) demonstrated a significant reduction in numbers of Renshaw interneurons in transgenic animals compared to age-matched control animals (p=0.001, Mann-Whitney Rank Sum test).

At 90 day of age, Renshaw interneurons mean count in transgenic animals (Figure 9B) was 2.11 per ventral horn (n=6, S.D=1.54, 712 ventral horns analyzed) compared to age-matched control animals (Figure 9A) mean count of 3.20 per ventral horn (n=6, S.D=1.71, 608 ventral horns analyzed). Statistical analysis (Figure 9C) illustrated a significant reduction in numbers of Renshaw interneurons in transgenic animals compared to age-matched control animals (p=0.001, Mann-Whitney Rank Sum test, power of the test, power of test=0.8).

At 110 day of age, Renshaw interneurons mean count in transgenic animals (Figure 11B) was 1.99 per ventral horn (n=6, S.D=1.42, 678 ventral horn analyzed), compared to age-matched control animals (Figure 11A) mean count of 2.90 per ventral horn (n=6, S.D=1.70, 614 ventral horn analyzed). Statistical analysis (Figure 11C) demonstrated a significant reduction in numbers of Renshaw interneurons in transgenic animals compared to age-matched control animals (p=0.001, Mann-Whitney Rank Sum test, power of the test).

At 148 day of age, Renshaw interneurons mean count in transgenic animals (Figure 13B) was 1.45 per ventral horn (n=6, S.D=1.41, 584 ventral horn analyzed), compared to age-matched control animals (Figure 13A) mean count of 2.96 per ventral horn (n=6, S.D=1.66, 502 ventral horn analyzed). Statistical analysis (Figure 13C) demonstrated a significant reduction in numbers of Renshaw interneurons in transgenic animals compared to age-matched control animals (p=0.001, Mann-Whitney Rank Sum test).

Result data demonstrates gephyrin and calbindin D28k immunoreactive putative Renshaw interneurons were impacted in G93A-SOD1 transgenic ALS-mouse model, as the numbers of putatively identified Renshaw interneurons in transgenic ALS-mouse model are significantly less than the age-matched control animals at five different timelines examined during this study (40, 70, 90, 110, 148 days of age); however, 28 days old G93A-SOD1 transgenic animals demonstrated a Renshaw interneurons count comparable to the age-matched control animals. Gephyrin and calbindin D28K immunoreactive Renshaw interneurons loss observed initially at 40 days old transgenic animals, as animals at this age demonstrated 36.07% less Renshaw interneurons than age-match control animals.

Statistical analysis demonstrated no significant reduction or increase in number of Renshaw interneurons among G93A-SOD1 transgenic animals aged 40, 70, 90, 110 days old (p=0.15, ANOVA on Ranks). While animals aged 148 days old demonstrated a significant reduction in number of Renshaw interneurons, compared to transgenic animals aged 40, 70, 90, 110 days old (p=0.001, ANOVA on Ranks test). (Figure 23)

Motor neurons count:

Every ninth lumbar spinal cord transverse section, processed with Toluidine blue for Nissl histochemistry, was utilized for the purpose of motor neurons enumeration. Only motor neurons displaying central profiles (clear nucleus and prominent nucleoli) were enumerated. Furthermore, the utilization of the neuron size criteria by image analysis with image j software (counting only neurons more that 35mm in diameter) enable us to exclude ϒ-neurons from our result ( The data are representative of only α-motor neurons number in Lumbar spinal cord ventral horns).

At 40 day of age (Figure 6), α-motor neurons mean count per ventral horn were: ALS-animals=11.45 (n=3, S.D=3.24, 58 ventral horns analyzed), compared to age-matched control animals count of 11.71 (n=3, S.D=3.84, 74 ventral horns analyzed). Statistical analysis (Figure 6C) demonstrated no significant reduction in numbers of α-motor neurons in transgenic animals compared to age-matched control animals (0.958, Mann-Whitney Rank Sum test, power of test=0.8)

At 70 day of age (Figure 8), α-motor neurons mean counts per ventral horn were: transgenic animals= 9.91 (n=3, S.D=4.18, 67 ventral horns analyzed), compared to 11.34 in age-matched control animals (n=3, S.D=3.03, 79 ventral horns analyzed). Statistical analysis demonstrated a significant reduction in numbers of α-motor neurons in transgenic animals compared to age-matched control animals (p=0.015, Mann-Whitney Rank Sum test, power of test=0.8)(Figure 8c).

At 90 days old animals (Figure 10), α-motor neurons mean counts per ventral horn were: 6.20 neurons (n=3, S.D=3.35, 79 ventral horns analyzed) in transgenic animals, compared to 12.10 neurons (n=3, S.D=4.40, 42 ventral horn analyzed) in control animals. Statistical analysis demonstrated a significant reduction in numbers of α-motor neurons in transgenic animals compared to age-matched control animals (p=<0.001, Mann-Whitney Rank Sum test, power of test=0.8)(Figure 10c).

At 110 days old animals (Figure 12), α-motor neurons mean counts per ventral horn were: 6.45 in transgenic animals (n=3, S.D=2.32, 71 ventral horns analyzed), compared to 11.56 in age-matched control animals (n=3, S.D=2.65, 75 ventral horn analyzed). Statistical analysis demonstrated a significant reduction in numbers of α-motor neurons in transgenic animals compared to age-matched control animals (p=<0.001, Mann-Whitney Rank Sum test, power of test=0.8)(Figure 12C)

At 148 days old animals (Figure 14), α-motor neurons counts per ventral horn were: 4.27 in transgenic animals (n=3, S.D=3.35, 63 ventral horn analyzed), compared to 12.62 in age-control animals (n=3, S.D=5.4, 69 ventral horn analyzed). Statistical analysis demonstrated a significant reduction in numbers of α-motor neurons in transgenic animals compared to age-matched control animals (p=<0.001, Mann-Whitney Rank Sum test, power of test=0.8)(Fig 14c).

Result data indicate a significant reduction in the number of α-motor neurons in lumbar spinal cord compared to control animals. The loss in motor neurons first noticed in transgenic animals aged 70 days, while 40 days old transgenic animals demonstrated no significant reduction in the numbers of α-motor neurons

Compared to age-matched control animals. Transgenic animals demonstrated a prominent cytoplasmic vacuolation of motor neuron somata, with no similar vacuolation in control animals. This cytoplasmic vacuolation illustrated in transgenic animals aged 90, 110, 148 days old (fig 10b &12b). The cytoplasmic Vacuolation noticed in some motor neurons in animals 90 days old, with difficulty; however, cytoplasmic vacuolation illustrated very clearly and more diffusely involved undying motor neurons in advanced age, symptomatic animals (110 and 148 days of age). Also, a prominent histological feature of gliosis (Figure 14b) in the ventral horn (small darkly stained cells and nuclei) observed in the lumbar spinal cord sections in advanced transgenic symptomatic animals (110, 148 days old animals), with no similar finding in age-matched control animals.

Result data confirms the motor neuron loss in the G93A-SOD1 mouse model utilized for this study. The loss in motor neuron noticed at animals aged 70 days old. At 40 days of age, there was no significant loss in the motor neuron in transgenic animals compared to age-matched control animals.

Analyzing control animals, from different ages (40, 70, 90, 110, 148), among each other demonstrated no significant inter-age variability in the number of α-motor neurons (p=0.88, ANOVA on Ranks). Analysis of transgenic animals from different ages demonstrated significant progressive reduction of motor neurons with advancement in age (p=<0.001, ANOVA on Ranks).

Superoxide dismutase localization in Renshaw interneuron:

Lumbar spinal cord tissue, from fifteen transgenic and age matched control animals, was immunolabeled with anti-gephyrin and anti-SOD1 antibody. The control non-transgenic littermate tissue demonstrated SOD1-labeling phenotype of minimal labeling intensity of the neuropile and white matter of the lumbar spinal cord (Figure 15A) and low intensity cytoplasmic labeling and strong perinuclear signal in putatively identified gephyrin-reactive Renshaw interneurons (Figure 16A, 16B, 16C), as well as, motor neurons (Figure 16D, 16E, 16F) in all of

A

Bthe examined control animals tissue (n=15).

The transgenic animals (n=15) demonstrated different phenotypic distribution of SOD1 enzyme characterized by increased intensity of labeling in the whole gray and white matter of the spinal cord (Figure 15B) with high intensity cytoplasmic labeling of the Renshaw interneurons soma and axon (Fig 17A, 17B, 17C), as well as, motor neurons (Fig 17D, 17E, 17F). The phenotypic distribution of SOD1 labeling of identified Renshaw interneurons was similar to that of motor neurons SOD1 phenotype in all of the examined transgenic and control animals.

Cleaved Caspase-3 co-localization with Renshaw interneuron:

Gephyrin Immunolabeled transverse lumbar spinal cord sections from transgenic and age-matched control animals at different timelines (28, 40, 70, 90, 110, 148 days of age) were utilized to asses the co-localization of cleaved caspase-3, an apoptosis marker, with the putatively identified Renshaw interneurons.

At 28 days of age, 74 Renshaw interneurons (n=3) were analyzed from transgenic animals compared to 88 Renshaw interneurons (n=3) from age- matched control animals. At 40 days of age; 130 Renshaw interneuron were analyzed from transgenic animals (n=3) compared to 148 cells from age-matched control animals (n=3). At 70 days old age; 107 Renshaw cells analyzed from transgenic animals (n=3) compared to 110 cells from age-matched control animals (n=3). At 90 days of age; 163 cells were analyzed from transgenic animals (n=4) compared to 181 Renshaw interneuron from age matched control animals (n=4). At 110 days of age; 132 Renshaw interneuron from transgenic animals compared to 95 Renshaw interneurons from age-matched control animals (n=3). At 148 days of age: 83 Renshaw interneurons were analyzed from transgenic animals compared to 83 cells from age -matched control animals.

Cleaved caspase-3 co-localized in only eleven gephyrin immunoreactive putatively identified Renshaw interneurons in one seventy-days old transgenic animal (Figure 15C&D). No other animals demonstrated a co-localization of cleaved caspase-3 with gephyrin reactive Renshaw interneurons.

Cytochrome c oxidase activity of the Renshaw interneurons.

Gephyrin and calbindin D28K immunohistochemically labeled lumbar spinal cord sections from transgenic and age-matched control animals at different representative ages (28, 40, 70, 90, 110, 148 days of age) were histochemically processed for cytochrome c activity reaction. The characteristic-immunolabeling pattern of Renshaw interneurons (dense, large gephyrin clusters and strong calbindin D28k labeling) was still detectable, in spite of the sequential method of labeling used for detection of cytochrome c reactivity. Putatively identified Renshaw interneurons were further analyzed for the purpose of qualitative assessment of cytochrome c enzyme reaction in the soma of those interneurons. Renshaw interneurons were categorized into lightly, moderately and deeply reacted cells (Figure 17). No Renshaw interneuron demonstrating deep cytochrome c reaction was observed in all of the analyzed transgenic and control animals tissue; Therefore, data were arranged into two categories only: Cytochrome c labeled Renshaw interneurons (included the moderately labeled neurons) and Cytochrome c unlabeled Renshaw interneurons (included lightly labeled and unlabeled Renshaw interneurons).

At 28 days of age: analysis of transgenic G93A-SOD1 animals demonstrated 92.07% of the putatively identified Renshaw interneurons with light or no cytochrome c reaction product and only 7.92% demonstrated moderate level of cytochrome c reaction product (n=3, 101 Renshaw interneuron analyzed). In age-matched control animals: 90.09% of the putatively identified Renshaw interneurons demonstrated light or no cytochrome c reaction product, while remaining 9.91% of Renshaw interneurons demonstrated moderate level of cytochrome c reaction product (n=3, 111 Renshaw interneurons analyzed). Statistical analysis, using Mann-Whitney Rank Sum test, revealed no significant difference in cytochrome c product reaction between transgenic and age-matched control animals (p=0..803, power of test= 0.8).

At 40 days of age; analysis of transgenic G93A-SOD1 animals demonstrated; 95.16% of the putatively identified Renshaw interneurons with light or no cytochrome c reaction product and only 4.83% demonstrated moderate level of cytochrome c reaction product (n=3, 124 Renshaw interneuron analyzed). In age-matched control animals: 88.98% of the putatively identified Renshaw interneurons demonstrated light or no cytochrome c reaction product, while remaining 11.02% of Renshaw interneurons demonstrated moderate level of cytochrome c reaction product (n=3, 127 Renshaw interneurons analyzed). Statistical analysis, using Mann-Whitney Rank Sum test, revealed no significant difference in cytochrome c product reaction between transgenic and age-matched control animals (p=0..909, power of test =0.8).

At 70 days of age: analysis of transgenic G93A-SOD1 animals demonstrated; 94.39% of the putatively identified Renshaw interneurons with light or no cytochrome c reaction product and only 5.61% demonstrated moderate level of cytochrome c reaction product (n=3, 107 Renshaw interneuron analyzed). In age-matched control animals: 90.52% of the putatively identified Renshaw interneurons demonstrated light or no cytochrome c reaction product, while remaining 9.48% of Renshaw interneurons demonstrated moderate level of cytochrome c reaction product (n=3, 116 Renshaw interneurons analyzed). Statistical analysis, using Mann-Whitney Rank Sum test, revealed no significant difference in cytochrome c product reaction between transgenic and age-matched control animals (p=0.617, power of test 0.8).

At 90 days of age: analysis of transgenic G93A-SOD1 animals demonstrated; 96.42% of the putatively identified Renshaw interneurons with light or no cytochrome c reaction product and only 3.57% demonstrated moderate level of cytochrome c reaction product (n=3, 112 Renshaw interneuron analyzed). In age-matched control animals: 94.62% of the putatively identified Renshaw interneurons demonstrated light or no cytochrome c reaction product, while remaining 5.38% of Renshaw interneurons demonstrated moderate level of cytochrome c reaction product (n=3, 130 Renshaw interneurons analyzed). Statistical analysis, using Mann-Whitney Rank Sum test, revealed no significant difference in cytochrome c product reaction between transgenic and age-matched control animals (p=0.808, power of test 0.8).

At 110 days of age: analysis of transgenic G93A-SOD1 animals demonstrated; 94.79% of the putatively identified Renshaw interneurons with light or no cytochrome c reaction product and only 5.21% demonstrated moderate level of cytochrome c reaction product (n=3, 96 Renshaw interneuron analyzed). In age-matched control animals: 93.64% of the putatively identified Renshaw interneurons demonstrated light or no cytochrome c reaction product, while remaining 7.29% of Renshaw interneurons demonstrated moderate level of cytochrome c reaction product (n=3, 110 Renshaw interneurons analyzed). Statistical analysis, using Mann-Whitney Rank Sum test, revealed no significant difference in cytochrome c product reaction between transgenic and age-matched control animals (p=0.887, power of test 0.8).

At 148 days of age: analysis of transgenic G93A-SOD1 animals demonstrated; 94.51% of the putatively identified Renshaw interneurons with light or no cytochrome c reaction product and only 5.49% demonstrated moderate level of cytochrome c reaction product (n=3, 91 Renshaw interneuron analyzed). In age-matched control animals: 92.81% of the putatively identified Renshaw interneurons demonstrated light or no cytochrome c reaction product, while remaining 7.19% of Renshaw interneurons demonstrated moderate level of cytochrome c reaction product (n=3, 139 Renshaw interneurons analyzed). Statistical analysis, using Mann-Whitney Rank Sum test, revealed no significant difference in cytochrome c product reaction bet

ween transgenic and age-matched control animals (p=0.823, power of test 0.8).

There was no significant reduction or increase in among animals from all controls age groups (One Way ANOVA on Ranks, p=0.532). Also there was no significant change in the cytochrome c activity reaction among all ALS-animals (One Way ANOVA on Ranks, p=0.135).

Results demonstrated there is no significant reduction or increase in the activity of cytochrome c enzyme in the identified gephyrin and calbindin D28K reactive Renshaw interneurons in transgenic G93A-SOD1 mice (Figure 18a, b), compared to age-matched control animals(Figure18c, d) in all of the examined ages (28, 40, 70, 90, 110, 148 days old) (Figure 18a, b, c, d). (Figure 23)

NADPH-diaphorase reaction in putatively identified Renshaw interneurons:

Gephyrin and calbindin D28K immunoreactive lumbar spinal cord sections were further processed to qualitatively assess the level of NADPH-diaphorase (NADPHd) reactivity in putatively identified Renshaw interneurons. The sequential staining method didn't dramatically impair the identification of gephyrin and calbindin D28K labeled Renshaw interneurons. NADPH-diaphoras reaction product of the putatively identified Renshaw interneurons was assessed qualitatively and described as (no or light labeling, moderately labeling, deeply labeled neurons)(Figure 20) .No putatively identified Renshaw interneuron demonstrated a dark NADPH-Diaphorase enzyme reaction product labeling; therefore, data was arranged into 2 categories: labeled interneurons (moderate NADPH-diaphorase labeling) and unlabeled interneurons (no or light NADPH-diaphorase reactivity)

At 28 days of age; 100% of putatively identified Renshaw interneurons demonstrated light cytoplasmic level of NADPHd reaction product in transgenic animals (n=3, 68 Renshaw interneuron analyzed), compared to control animals in which 100% of the identified renshaw interneurons demonstrated lightly NADPHd activity (n=3, 73 Renshaw interneurons analyzed). Statistical analysis demonstrated no significant reduction or increase in the activity of NADPH-diaphorase reaction (p=0.803, Mann-Whitney Rank Sum test, power of test=0.8).

At 40 days of age; 100% of the putatively identified Renshaw interneurons in transgenic G93A-SOD1 animals demonstrated light NADPHd reaction (n=3, 91 Renshaw interneurons analyzed), Compared to age-matched control animals; 99.11% of putatively identified Renshaw interneurons demonstrated light NADPHd labeling and only 0.88% of Renshaw interneurons demonstrated moderate NADPHd reaction product (n=3, 113 Renshaw interneurons analyzed). Statistical analysis demonstrated no significant reduction or increase in the activity of NADPH-diaphorase reaction (p=0.909, Mann-Whitney Rank Sum test, power of test=0.8)

At 70 days of age; 100% of the putatively identified Renshaw interneurons demonstrated light NADPHd reaction product in transgenic animals (n=3, 89 Renshaw interneurons analyzed). Compared to age-matched control animals; 98.76% demonstrated light NADPHd reaction product and only 1.23% of the identified Renshaw interneurons demonstrated moderate NADPHd reaction product (n=3, 81 Renshaw interneurons analyzed). Statistical analysis demonstrated no significant reduction or increase in the activity of NADPH-diaphorase reaction (p=0.617, Mann-Whitney Rank Sum test, power of test=0.8)

At 90 days of age, 100% of the identified Renshaw interneurons demonstrated light NADPHd labeling in transgenic animals (n=3, 86 Renshaw interneurons analyzed). Compared to control animals; 99.03% of the identified renshaw interneurons with light NADPHd reactivity and only 0.96% moderately labeled with NADPHd (n=3, 104 Renshaw interneurons analyzed). Statistical analysis demonstrated no significant reduction or increase in the activity of NADPH-diaphorase reaction (p=0.808, Mann-Whitney Rank Sum test, power of test=0.8)

At 110 days of age, 99% of the putatively identified Renshaw interneurons demonstrated light NADPHd reaction product in transgenic animals and only 1% demonstrated moderate NADPHd reactivity (n=3, 94 Renshaw interneurons analyzed). Compared to age-matched control animals; 99.05% demonstrated light NADPHd reaction and only 0.94% of the identified Renshaw interneurons demonstrated moderate NADPHd histochemical labeling (n=3, 106 Renshaw interneurons analyzed). Statistical analysis demonstrated no significant reduction or increase in the activity of NADPH-diaphorase reaction (p=0.887, Mann-Whitney Rank Sum test, power of test=0.8)

At 148 days of age; 100% of the putatively identified Renshaw interneurons demonstrated light NADPHd reaction product in transgenic animals (n=3, 92 Renshaw interneurons analyzed). Compared to age-matched control animals; 100% also demonstrated light NADPHd histochemical labeling (n=3, 127 Renshaw interneurons analyzed). Statistical analysis demonstrated no significant reduction or increase in the activity of NADPH-diaphorase reaction (p=0.823, Mann-Whitney Rank Sum test, power of test=0.8).

Result demonstrates no significant reduction or increase in the activity of NADPH diaphorase in the identified gephyrin and calbindin D28K reactive Renshaw interneurons in transgenic G93A-SOD1 mice (Figure 21a, b), compared to age-matched control animals (Figure 21c, d) in all of the examined ages (28, 40, 70, 90, 110, 148 days old) (Figure 21a, b, c, d).

Fig 20

Summary.

Renshaw interneurons count.

Renshaw interneurons enumeration in ALS and control animals in 6 representative ages (28, 40, 70, 90, 110, 148 days old) demonstrated a significant reduction in the numbers of Renshaw interneurons in transgenic animals compared to age-matched control animals in 5 different age timelines: 40, 70, 90, 110, 148 days old animals. Renshaw interneurons loss was observed in ALS-animals during the early pre-symptomatic period (40 day of age). There was a 36.7% loss of Renshaw interneurons in ALS-Animals compared to age-matched control animals aged 40 days old. impaction of those interneurons continued during the subsequent ages (70, 90, 110, 148 days old animals). Statistical analysis demonstrated that the loss of Renshaw interneurons was not progressive until after the age of 110 days where the count Renshaw interneurons in ALS-animals was 51.01% less than in age-matched control animals

(Fig .22)

Motor neurons count.

Enumeration of motor neurons in transgenic and age matched control animals aged 40, 70, 90, 110, 148 days old enabled us to timeline the motor neuron loss in this transgenic animal model, providing a comparable data with timeline of Renshaw interneurons loss. The significant reduction in motor neurons count in ALS animals observed during 70 day of age, nearly a month after detectable loss of Renshaw interneurons in this Transgenic mouse model.(Fig 23). Loss of motor neurons was progressive over the examined age-timeline

SOD1 localization in Renshaw interneurons.

The similarities between Motor neurons and Renshaw interneurons phenotypic distribution in intra, as well as extra, neuronal localization of SOD1 enzyme between transgenic and control animals provided the conclusion that Renshaw interneurons do have increased level of SOD1, specifically mutant SOD1. Which is the hallmark of G93A-SOD1 transgenic mouse model.

Cytochrome c oxidase activity.

Qualitative assessment of cytochrome c oxidase histochemical labeling of Renshaw interneurons didn't demonstrate any changes in the quality of labeling of Renshaw interneuron between Transgenic and age-matched control animals. (Figure 23)

NADPH-diaphorase activity.

Qualitative assessment of NADPH diaphorase histochemical labeling of Renshaw interneurons didn't demonstrate any changes in the quality of NADPH diaphorase reaction product within Renshaw interneurons between Transgenic and age-matched control animals.(Fig 24)

Cleaved caspase-3 localization with Renshaw interneurons.

The detection of Renshaw interneurons labeling with cleaved caspase-3 in one of the 70 days old transgenic animals provides the evidence that apoptosis might play a role in Renshaw interneurons impaction in G93A-SOD1 transgenic animals.

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