Browsing by Author "Walker, Chandler L."

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    Adipose-derived stem cell conditioned medium for the treatment of amyotrophic lateral sclerosis: pre-clinical evidence and potential for clinical application
    (Medknow Publications, 2019-09) Walker, Chandler L.; Department of Biomedical and Applied Sciences, School of Dentistry
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    Adipose-derived stem cell conditioned medium impacts asymptomatic peripheral neuromuscular denervation in the mutant superoxide dismutase (G93A) transgenic mouse model of amyotrophic lateral sclerosis
    (IOS, 2018-09) Walker, Chandler L.; Meadows, Rena M.; Merfeld-Clauss, Stephanie; Du, Yansheng; March, Keith L.; Jones, Kathryn J.; Biomedical Sciences and Comprehensive Care, School of Dentistry
    Background:Amyotrophic lateral sclerosis (ALS) is devastating, leading to paralysis and death. Disease onset begins pre-symptomatically through spinal motor neuron (MN) axon die-back from musculature at ∼47 days of age in the mutant superoxide dismutase 1 (mSOD1G93A) transgenic ALS mouse model. This period may be optimal to assess potential therapies. We previously demonstrated that post-symptomatic adipose-derived stem cell conditioned medium (ASC-CM) treatment is neuroprotective in mSOD1G93A mice. We hypothesized that early disease onset treatment could ameliorate neuromuscular junction (NMJ) disruption. Objective:To determine whether pre-symptom administration of ASC-CM prevents early NMJ disconnection. Methods:We confirmed the NMJ denervation time course in mSOD1G93A mice using co-labeling of neurofilament and post-synaptic acetylcholine receptors (AchR) by α-bungarotoxin. We determined whether ASC-CM ameliorates early NMJ loss in mSOD1G93A mice by systemically administering 200μl ASC-CM or vehicle medium daily from post-natal days 35 to 47 and quantifying intact NMJs through co-labeling of neurofilament and synaptophysin with α-bungarotoxin in gastrocnemius muscle. Results:Intact NMJs were significantly decreased in 47 day old mSOD1G93A mice (p < 0.05), and daily systemic ASC-CM prevented disease-induced NMJ denervation compared to vehicle treated mice (p < 0.05). Conclusions:Our results lay the foundation for testing the long-term neurological benefits of systemic ASC-CM therapy in the mSOD1G93A mouse model of ALS.
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    Biphasic bisperoxovanadium administration and Schwann cell transplantation for repair after cervical contusive spinal cord injury
    (Elsevier, 2015-02) Walker, Chandler L.; Wang, Xiaofei; Bullis, Carli; Liu, Nai-Kui; Lu, Qingbo; Fry, Colin; Deng, Lingxiao; Xu, Xiao-Ming; Department of Neurological Surgery, IU School of Medicine
    Schwann cells (SCs) hold promise for spinal cord injury (SCI) repair; however, there are limitations for its use as a lone treatment. We showed that acute inhibition of the phosphatase and tensin homolog deleted on chromosome ten (PTEN) by bisperoxovanadium (bpV) was neuroprotective and enhanced function following cervical hemicontusion SCI. We hypothesized that combining acute bpV therapy and delayed SC engraftment would further improve neuroprotection and recovery after cervical SCI. Adult female Sprague-Dawley (SD) rats were randomly sorted into 5 groups: sham, vehicle, bpV, SC transplantation, and bpV+SC transplantation. SCs were isolated from adult green fluorescent protein (GFP)-expressing SD rats (GFP-SCs). 200 μg/kg bpV(pic) was administered intraperitoneally (IP) twice daily for 7 days post-SCI in bpV-treated groups. GFP-SCs (1×10(6) in 5 μl medium) were transplanted into the lesion epicenter at the 8th day post-SCI. Forelimb function was tested for 10 weeks and histology was assessed. bpV alone significantly reduced lesion (by 40%, p<0.05) and cavitation (by 65%, p<0.05) and improved functional recovery (p<0.05) compared to injury alone. The combination promoted similar neuroprotection (p<0.01 vs. injury); however, GFP-SCs alone did not. Both SC-transplanted groups exhibited remarkable long-term SC survival, SMI-31(+) axon ingrowth and RECA-1(+) vasculature presence in the SC graft; however, bpV+SCs promoted an 89% greater axon-to-lesion ratio than SCs only. We concluded that bpV likely contributed largely to the neuroprotective and functional benefits while SCs facilitated considerable host-tissue interaction and modification. The combination of the two shows promise as an attractive strategy to enhance recovery after SCI.
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    Bisperoxovanadium Mediates Neuronal Protection through Inhibition of PTEN and Activation of PI3K/AKT-mTOR Signaling after Traumatic Spinal Injuries
    (Mary Ann Liebert, Inc., publishers, 2019-08-30) Walker, Chandler L.; Wu, Xiangbing; Liu, Nai-Kui; Xu, Xiao-Ming; Neurological Surgery, School of Medicine
    Although mechanisms involved in progression of cell death in spinal cord injury (SCI) have been studied extensively, few are clear targets for translation to clinical application. One of the best-understood mechanisms of cell survival in SCI is phosphatidylinositol-3-kinase (PI3K)/Akt and associated downstream signaling. Clear therapeutic efficacy of a phosphatase and tensin homologue (PTEN) inhibitor called bisperoxovanadium (bpV) has been shown in SCI, traumatic brain injury, stroke, and other neurological disease models in both neuroprotection and functional recovery. The present study aimed to elucidate mechanistic influences of bpV activity in neuronal survival in in vitro and in vivo models of SCI. Treatment with 100 nM bpV(pic) reduced cell death in a primary spinal neuron injury model (p < 0.05) in vitro, and upregulated both Akt and ribosomal protein S6 (pS6) activity (p < 0.05) compared with non-treated injured neurons. Pre-treatment of spinal neurons with a PI3K inhibitor, LY294002 or mammalian target of rapamycin (mTOR) inhibitor, rapamycin blocked bpV activation of Akt and ribosomal protein S6 activity, respectively. Treatment with bpV increased extracellular signal-related kinase (Erk) activity after scratch injury in vitro, and rapamycin reduced influence by bpV on Erk phosphorylation. After a cervical hemicontusive SCI, Akt phosphorylation decreased in total tissue via Western blot analysis (p < 0.01) as well as in penumbral ventral horn motor neurons throughout the first week post-injury (p < 0.05). Conversely, PTEN activity appeared to increase over this period. As observed in vitro, bpV also increased Erk activity post-SCI (p < 0.05). Our results suggest that PI3K/Akt signaling is the likely primary mechanism of bpV action in mediating neuroprotection in injured spinal neurons.
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    Bisperoxovanadium promotes motor neuron survival and neuromuscular innervation in amyotrophic lateral sclerosis
    (BMC, 2021-10-11) Wang, Junmei; Tierney, Lydia; Mann, Ranjeet; Lonsway, Thomas; Walker, Chandler L.; Biomedical Sciences and Comprehensive Care, School of Dentistry
    Amyotrophic lateral sclerosis (ALS) is the most common motor neuron (MN) disease, with no present cure. The progressive loss of MNs is the hallmark of ALS. We have previously shown the therapeutic effects of the phosphatase and tensin homolog (PTEN) inhibitor, potassium bisperoxo (picolinato) vanadium (bpV[pic]), in models of neurological injury and demonstrated significant neuroprotective effects on MN survival. However, accumulating evidence suggests PTEN is detrimental for MN survival in ALS. Therefore, we hypothesized that treating the mutant superoxide dismutase 1 G93A (mSOD1G93A) mouse model of ALS during motor neuron degeneration and an in vitro model of mSOD1G93A motor neuron injury with bpV(pic) would prevent motor neuron loss. To test our hypothesis, we treated mSOD1G93A mice intraperitoneally daily with 400 μg/kg bpV(pic) from 70 to 90 days of age. Immunolabeled MNs and microglial reactivity were analyzed in lumbar spinal cord tissue, and bpV(pic) treatment significantly ameliorated ventral horn motor neuron loss in mSOD1G93A mice (p = 0.003) while not significantly altering microglial reactivity (p = 0.701). Treatment with bpV(pic) also significantly increased neuromuscular innervation (p = 0.018) but did not affect muscle atrophy. We also cultured motor neuron-like NSC-34 cells transfected with a plasmid to overexpress mutant SOD1G93A and starved them in serum-free medium for 24 h with and without bpV(pic) and downstream inhibitor of Akt signaling, LY294002. In vitro, bpV(pic) improved neuronal viability, and Akt inhibition reversed this protective effect (p < 0.05). In conclusion, our study indicates systemic bpV(pic) treatment could be a valuable neuroprotective therapy for ALS.
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    Carboxyl-terminal modulator protein regulates Akt signaling during skeletal muscle atrophy in vitro and a mouse model of amyotrophic lateral sclerosis
    (Springer Nature, 2019-03-08) Wang, Junmei; Fry, Colin M. E.; Walker, Chandler L.; Biomedical Sciences and Comprehensive Care, School of Dentistry
    Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease involving motor neuron death, paralysis and, ultimately, respiratory failure. Motor neuron dysfunction leads to target skeletal muscle atrophy involving dysregulation of downstream cell survival, growth and metabolic signaling. Decreased Akt activity is linked to muscle atrophy in ALS and is associated with increased atrophy gene expression. Unfortunately, the regulating mechanism of Akt activity in atrophic muscle remains unclear. Recent research indicates a role of carboxyl-terminal modulator protein (CTMP) in Akt-signaling related neurologic dysfunction and skeletal muscle metabolism. CTMP is known to bind and reduce Akt phosphorylation and activation. We hypothesized that CTMP expression might progressively increase in ALS skeletal muscle as the disease progresses, downregulating Akt activity. We found that CTMP protein expression significantly increased in hindlimb skeletal muscle in the mSOD1G93A mouse model of ALS in late stages of the disease (P < 0.05), which negatively correlated with Akt phosphorylation over this period (R2 = -0.77). Co-immunoprecipitation of Akt revealed CTMP binding in pre-symptomatic and end-stage skeletal muscle, suggesting a possible direct role in reduced Akt signaling during disease progression. Inflammatory TNFα and downstream cellular degradation process markers for autophagy, lysosome production, and atrophy significantly increased in a pattern corresponding to increased CTMP expression and reduced Akt phosphorylation. In an in vitro model of skeletal muscle atrophy, differentiated C2C12 cells exhibited reduced Akt activity and decreased FOXO1 phosphorylation, a process known to promote transcription of atrophy genes in skeletal muscle. These results corresponded with increased Atrogin-1 expression compared to healthy control cells (P < 0.05). Transfection with CTMP siRNA significantly increased Akt phosphorylation in atrophic C2C12 cells, corresponding to significantly decreased CTMP expression. In conclusion, this is the first study to provide evidence for a link between elevated CTMP expression, downregulated Akt phosphorylation and muscle atrophy in ALS and clearly demonstrates a direct influence of CTMP on Akt phosphorylation in an in vitro muscle cell atrophy model.
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    Characterization and Modulation of PI3K-Akt Signaling Following Contusive SCI
    (Office of the Vice Chancellor for Research, 2011-04-08) Walker, Chandler L.; Risberg, Emelie C.; Zhu, Yusheng; Liu, Naikui; Xu, Xiao-Ming
    Spinal cord injury (SCI) is devastating, with most injuries being contusive/compressive injuries at the cervical spinal level. There are two mechanisms of damage after acute contusive SCI: a primary mechanical insult to the cord, and secondary injury induced by many biological events, including inflammation and signal-mediated cell death. The extent of tissue damage correlates with functional loss after SCI, therefore it is critical to protect neural tissue for preservation of functional ability. Focusing on cellular signaling events following SCI is a promising direction of investigation, as modulation of such pathways can promote neuroprotection or regeneration following injury. Two particular signaling pathways have been highlighted as mediators of cellular survival post-central nervous system (CNS) injury, the MEK-Erk and PI3K-Akt pathways. Reducing Erk activity has been shown to promote neuroprotection and reduced reactive gliosis, while reduction of PI3K-Akt signaling likely results in initiation of cellular death. Recent studies have demonstrated promotion of regrowth of adult corticospinal (CST) neurons and protection of motor neuron atrophy by disinhibition of PI3K via PTEN deletion or knock-down in these cells. Understanding the signal pathways and mechanisms involved in different cell types, when such response occurs, and the potential interaction between pathways is essential for maximizing development of optimal approaches to treatment following SCI. This study highlights PI3K-Akt signaling involvement following injury, with future directions aimed at better understanding this pathway for targeting therapies to mediate anatomical and functional preservation and recovery following SCI.
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    Facial nerve repair utilizing intraoperative repair strategies
    (Wiley, 2020-05-28) Brown, Brandon L.; Sandelski, Morgan M.; Drejet, Sarah M.; Runge, Elizabeth M.; Shipchandler, Taha Z.; Jones, Kathryn J.; Walker, Chandler L.; Radiology and Imaging Sciences, School of Medicine
    Objectives To determine whether functional and anatomical outcomes following suture neurorrhaphy are improved by the addition of electrical stimulation with or without the addition of polyethylene glycol (PEG). Methods In a rat model of facial nerve injury, complete facial nerve transection and repair was performed via (a) suture neurorrhaphy alone, (b) neurorrhaphy with the addition of brief (30 minutes) intraoperative electrical stimulation, or (c) neurorrhaphy with the addition electrical stimulation and PEG. Functional recovery was assessed weekly for 16 weeks. At 16 weeks postoperatively, motoneuron survival, amount of regrowth, and specificity of regrowth were assessed by branch labeling and tissue analysis. Results The addition of brief intraoperative electrical stimulation improved all functional outcomes compared to suturing alone. The addition of PEG to electrical stimulation impaired this benefit. Motoneuron survival, amount of regrowth, and specificity of regrowth were unaltered at 16 weeks postoperative in all treatment groups. Conclusion The addition of brief intraoperative electrical stimulation to neurorrhaphy in this rodent model shows promising neurological benefit in the surgical repair of facial nerve injury. Level of Evidence Animal study.
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    Functional and Anatomical Outcomes of Facial Nerve Injury With Application of Polyethylene Glycol in a Rat Model
    (American Medical Association, 2019-01-01) Brown, Brandon L.; Asante, Tony; Welch, Haley R.; Sandelski, Morgan M.; Drejet, Sarah M.; Shah, Kishan; Runge, Elizabeth M.; Shipchandler, Taha Z.; Jones, Kathryn J.; Walker, Chandler L.; Anatomy and Cell Biology, IU School of Medicine
    Importance: Functional and anatomical outcomes after surgical repair of facial nerve injury may be improved with the addition of polyethylene glycol (PEG) to direct suture neurorrhaphy. The application of PEG has shown promise in treating spinal nerve injuries, but its efficacy has not been evaluated in treatment of cranial nerve injuries. Objective: To determine whether PEG in addition to neurorrhaphy can improve functional outcomes and synkinesis after facial nerve injury. Design, Setting, and Subjects: In this animal experiment, 36 rats underwent right facial nerve transection and neurorrhaphy with addition of PEG. Weekly behavioral scoring was done for 10 rats for 6 weeks and 14 rats for 16 weeks after the operations. In the 16-week study, the buccal branches were labeled and tissue analysis was performed. In the 6-week study, the mandibular and buccal branches were labeled and tissue analysis was performed. Histologic analysis was performed for 10 rats in a 1-week study to assess the association of PEG with axonal continuity and Wallerian degeneration. Six rats served as the uninjured control group. Data were collected from February 8, 2016, through July 10, 2017. Intervention: Polyethylene glycol applied to the facial nerve after neurorrhaphy. Main Outcomes and Measures: Functional recovery was assessed weekly for the 16- and 6-week studies, as well as motoneuron survival, amount of regrowth, specificity of regrowth, and aberrant branching. Short-term effects of PEG were assessed in the 1-week study. Results: Among the 40 male rats included in the study, PEG addition to neurorrhaphy showed no functional benefit in eye blink reflex (mean [SEM], 3.57 [0.88] weeks; 95% CI, -2.8 to 1.9 weeks; P = .70) or whisking function (mean [SEM], 4.00 [0.72] weeks; 95% CI, -3.6 to 2.4 weeks; P = .69) compared with suturing alone at 16 weeks. Motoneuron survival was not changed by PEG in the 16-week (mean, 132.1 motoneurons per tissue section; 95% CI, -21.0 to 8.4; P = .13) or 6-week (mean, 131.1 motoneurons per tissue section; 95% CI, -11.0 to 10.0; P = .06) studies. Compared with controls, neither surgical group showed differences in buccal branch regrowth at 16 (36.9 motoneurons per tissue section; 95% CI, -14.5 to 22.0; P = .28) or 6 (36.7 motoneurons per tissue section; 95% CI, -7.8 to 18.5; P = .48) weeks or in the mandibular branch at 6 weeks (25.2 motoneurons per tissue section; 95% CI, -14.5 to 15.5; P = .99). Addition of PEG had no advantage in regrowth specificity compared with suturing alone at 16 weeks (15.3% buccal branch motoneurons with misguided projections; 95% CI, -7.2% to 11.0%; P = .84). After 6 weeks, the number of motoneurons with misguided projections to the mandibular branch showed no advantage of PEG treatment compared with suturing alone (12.1% buccal branch motoneurons with misguided projections; 95% CI, -8.2% to 9.2%; P = .98). In the 1-week study, improved axonal continuity and muscular innervation were not observed in PEG-treated rats. Conclusions and Relevance: Although PEG has shown efficacy in treating other nervous system injuries, PEG in addition to neurorraphy was not beneficial in a rat model of facial nerve injury. The addition of PEG to suturing may not be warranted in the surgical repair of facial nerve injury. Level of Evidence: NA.
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    Functional and Histological Gender Comparison of Age-Matched Rats after Moderate Thoracic Contusive Spinal Cord Injury
    (Mary Ann Liebert, 2019-05-28) Walker, Chandler L.; Fry, Colin M.E.; Wang, Junmei; Du, Xiaolong; Zuzzio, Kirstin; Liu, Nai-Kui; Walker, Melissa J.; Xu, Xiao-Ming; Neurological Surgery, School of Medicine
    Spinal cord injury (SCI) afflicts hundreds of thousands of Americans, and most SCI (∼80%) occurs in males. In experimental animal models, however, many studies used females. Funding agencies like the National Institutes of Health recommend that new proposed studies should include both genders due to variations in gender response to injuries, diseases, and treatments. However, cost and considerations for some animal models, such as SCI, affect investigators in adapting to this recommendation. Research has increased comparing gender effects in various disease and injury models, including SCI. However, most studies use weight-matched animals, which poses issues in comparing results and outcomes. The present study compared histologic and functional outcomes between age-matched male and female Sprague-Dawley rats in a moderate thoracic contusion SCI model. Cresyl violet and eosin staining showed no significant differences in lesion volume between genders after 9 weeks post-SCI (p > 0.05). Luxol fast blue–stained spared myelin was similar between genders, although slightly greater (∼6%) in spared myelin, compared with cord volume (p = 0.044). Glial reactivity and macrophage labeling in the lesion area was comparable between genders, as well. Basso, Beattie, Bresnahan (BBB) functional scores were not significantly different between genders, and Hargreaves thermal hyperalgesia and Gridwalk sensorimotor analyses also were similar between genders, compared with uninjured gender controls. Analysis of covariance showed weight did not influence functional recovery as assessed through BBB (p = 0.65) or Gridwalk assessment (p = 0.63) in this study. In conclusion, our findings suggest age-matched male and female rats recover similarly in a common clinically relevant SCI model.