Ching Yi Lin

Nerve Regeneration Restores Supraspinal Control of Bladder Function after Complete Spinal Cord Injury

A life-threatening disability after complete spinal cord injury is urinary dysfunction, which is attributable to lack of regeneration of supraspinal pathways that control the bladder. Although numerous strategies have been proposed that can promote the regrowth of severed axons in the adult CNS, at present, the approaches by which this can be accomplished after complete cord transection are quite limited. In the present study, we modified a classic peripheral nerve grafting technique with the use of chondroitinase to facilitate the regeneration of axons across and beyond an extensive thoracic spinal cord transection lesion in adult rats. The novel combination treatment allows for remarkably lengthy regeneration of certain subtypes of brainstem and propriospinal axons across the injury site and is followed by markedly improved urinary function. Our studies provide evidence that an enhanced nerve grafting strategy represents a potential regenerative treatment after severe spinal cord injury.

Fibronectin Inhibits Chronic Pain Development after Spinal Cord Injury

Chronic pain following spinal cord injury (SCI) is a highly prevalent clinical condition that is difficult to treat. Using both von Frey filaments and radiant infrared heat to assess mechanical allodynia and thermal hyperalgesia, respectively, we have demonstrated that a one-time injection of fibronectin (50 μg/mL) into the spinal dorsal column (1 μL/min each injection for a total of 5 μL) immediately after SCI inhibits the development of mechanical allodynia (but not thermal hyperalgesia) over an 8-month observation period following spinal cord dorsal column crush (DCC). DCC will only induce mechanical Allodynia, but not thermal hyperalgesia or overt motor deficits. By applying various fibronectin fragments as well as competitive inhibitors, these effects were shown to be dependent on the connecting segment-1 (CS-1) motif of fibronectin. Furthermore, we found that acute fibronectin treatment diminished inflammation and blood-spinal cord barrier permeability, which in turn leads to enhanced fiber sparing and sprouting. In particular, the reduction of serotonin (5-HT) in the superficial dorsal horn, an important descending brainstem system in the modulation of pain, was blocked with fibronectin treatment. We conclude that treatment of SCI with fibronectin preserves sensory regulation and prevents the development of chronic allodynia, providing a potential therapeutic intervention to treat chronic pain following SCI.

Expression of Suppressor of Cytokine Signaling-3 (SOCS3) and its role in neuronal death after complete spinal cord injury

The present study investigates the endogenous expression of Suppressor of Cytokine Signaling-3 (SOCS3) after spinal cord injury (SCI) and its effect on SCI-induced cell death in vivo. In addition, we determined whether a reduction of SOCS3 expression induced by microinjection of short hairpin RNA (shSOCS3) carried by lentivirus into spinal cord provides cellular protection after SCI. We demonstrated that complete transection of rat T8 spinal cord induced SOCS3 expression at the mRNA and protein levels as early as 2days post-injury, which was maintained up to 14days. SOCS3 immunoreactivity was detected in neurons and activated microglia after SCI. We also demonstrated that SCI induces phosphorylation of proteins that are involved in signal transduction and transcription-3 (STAT3) in neurons, which induced SOCS3 expression. Western blot analyses and double-immunofluorescent staining showed significant up-regulation of the pro-apoptotic protein Bax, increases in the ratio of Bax to the anti-apoptotic protein Bcl-2, and up-regulation of cleaved caspase-3 in neurons. Treatment with shSOCS3 inhibited SCI-induced mRNA expression of SOCS3 2days post-injury and suppressed SCI-induced Bax expression 7days after SCI, both rostral and caudal to the lesion. Moreover, treatment with shSOCS3 inhibited SCI-induced neuronal death and protected neuronal morphology both rostral and caudal to the injury site 7days post-injury. Our results suggest that the STAT3/SOCS3 signaling pathway plays an important role in regulating neuronal death after SCI.

Peripheral nerve transplantation combined with acidic fibroblast growth factor and chondroitinase induces regeneration and improves urinary function in complete spinal cord transected adult mice

The loss of lower urinary tract (LUT) control is a ubiquitous consequence of a complete spinal cord injury, attributed to a lack of regeneration of supraspinal pathways controlling the bladder. Previous work in our lab has utilized a combinatorial therapy of peripheral nerve autografts (PNG), acidic fibroblast growth factor (aFGF), and chondroitinase ABC (ChABC) to treat a complete T8 spinal cord transection in the adult rat, resulting in supraspinal control of bladder function. In the present study we extended these findings by examining the use of the combinatorial PNG+aFGF+ChABC treatment in a T8 transected mouse model, which more closely models human urinary deficits following spinal cord injury. Cystometry analysis and external urethral sphincter electromyograms reveal that treatment with PNG+aFGF+ChABC reduced bladder weight, improved bladder and external urethral sphincter histology, and significantly enhanced LUT function, resulting in more efficient voiding. Treated mice’s injured spinal cord also showed a reduction in collagen scaring, and regeneration of serotonergic and tyrosine hydroxylase-positive axons across the lesion and into the distal spinal cord. Regeneration of serotonin axons correlated with LUT recovery. These results suggest that our mouse model of LUT dysfunction recapitulates the results found in the rat model and may be used to further investigate genetic contributions to regeneration failure.

Neurotrophic factors rescue basal forebrain cholinergic neurons and improve performance on a spatial learning test

This study investigated whether animals sustaining experimental damage to the basal forebrain cholinergic system would benefit from treatment with exogenous neurotrophic factors. Specifically, we set out to determine whether neurotrophic factors would rescue damaged cholinergic neurons and improve behavioral performance on a spatial learning and memory task. Adult rats received bilateral injections of either saline (controls) or 192 IgG-saporin to damage basal forebrain cholinergic neurons (BFCNs). Two weeks later, animals received implants of an Alzet mini-pump connected to cannulae implanted bilaterally in the lateral ventricles. Animals received infusions of nerve growth factor (NGF), neurotrophin 3 (NT3), a combination of NGF and NT3, or a saline control over a 4-week period. Compared to saline-treated controls, animals sustaining saporin-induced damage to BFCNs took significantly more trials to learn a delayed match to position task and also performed more poorly on subsequent tests, with increasing delays between test runs. In contrast, animals infused with neurotrophins after saporin treatment performed significantly better than animals receiving saline infusions; no differences were detected for performance scores among animals infused with NGF, NT3, or a combination of NGF and NT3. Studies of ChAT immunnocytochemical labeling of BFCNs revealed a reduction in the numbers of ChAT-positive neurons in septum, nucleus of diagonal band, and nucleus basalis in animals treated with saporin followed by saline infusions, whereas animals treated with infusions of NGF, NT3 or a combination of NGF and NT3 showed only modest reductions in ChAT-positive neurons. Together, these data support the notion that administration of neurotrophic factors can rescue basal forebrain cholinergic neurons and improve learning and memory performance in rats.

Effects of Reducing Suppressors of Cytokine Signaling-3 (SOCS3) Expression on Dendritic Outgrowth and Demyelination after Spinal Cord Injury.

Suppressors of cytokine signaling-3 (SOCS3) is associated with limitations of nerve growth capacity after injury to the central nervous system. Although genetic manipulations of SOCS3 can enhance axonal regeneration after optic injury, the role of SOCS3 in dendritic outgrowth after spinal cord injury (SCI) is still unclear. The present study investigated the endogenous expression of SOCS3 and its role in regulating neurite outgrowth in vitro. Interleukin-6 (IL-6) induces SOCS3 expression at the mRNA and protein levels in neuroscreen-1 (NS-1) cells. In parallel to SOCS3 expression, IL-6 induced tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3) in NS-1 cells. Lentiviral delivery of short hairpin RNA (shSOCS3) (Lenti-shSOCS3) to decrease SOCS3 expression into NS-1 cells enhanced IL-6-induced tyrosine phosphorylation of STAT3 (P-STAT3 Tyr705) and promoted neurite outgrowth. In addition, we determined if reduction of SOCS3 expression by microinjection of Lenti-shSOCS3 into spinal cord enhances dendrite outgrowth in spinal cord neurons after SCI. Knocking down of SOCS3 in spinal cord neurons with Lenti-shSOCS3 increased complete SCI-induced P-STAT3 Tyr705. Immunohistochemical analysis showed that complete SCI induced a significant reduction of microtubule association protein 2-positive (MAP-2+) dendrites in the gray and white matter at 1 and 4 weeks after injury. The SCI-induced reduction of MAP-2+ dendrites was inhibited by infection with Lenti-shSOCS3 in areas both rostral and caudal to the lesion at 1 and 4 weeks after complete SCI. Furthermore, shSOCS3 treatment enhanced up-regulation of growth associated protein-43 (GAP-43) expression, which co-localized with MAP-2+ dendrites in white matter and with MAP-2+ cell bodies in gray matter, indicating Lenti-shSOCS3 may induce dendritic regeneration after SCI. Moreover, we demonstrated that Lenti-shSOCS3 decreased SCI-induced demyelination in white matter of spinal cord both rostral and caudal to the injury site 1 week post-injury, but not rostral to the injury at 4 weeks post-injury. Importantly, similar effects as Lenti-shSOCS3 on increasing MAP-2+ intensity and dendrite length, and preventing demyelination were observed when a second shSOCS3 (Lenti-shSOCS3 #2) was applied to rule out the possibilities of off target effects of shRNA. Collectively, these results suggest that knocking down of SOCS3 enhances dendritic regeneration and prevents demyelination after SCI.

Frequency-dependent effects of contralateral repetitive transcranial magnetic stimulation on penicillin-induced seizures

Transcranial magnetic stimulation (TMS) has been shown to modulate multiple brain functions, warranting further exploration in clinical applications. TMS treatment for epilepsy is particularly promising because of its anti-convulsive capabilities. However, TMS has been found to both inhibit and facilitate various experimental and clinical seizures, depending on the TMS parameters used. Repetitive TMS (rTMS) pulse frequency is recognized as one of the most influential parameters and thus was investigated in this study at 1, 5 and 10 Hz for its effects on a rat model of penicillin-induced seizures. High-dose penicillin-induced seizures were characterized by a combination of myoclonic and tonic-clonic (GTC) seizures. rTMS effects were analyzed with intracranial electroencephalographic (iEEG) data and video-captured behaviors. Animals treated with 1 and 5 Hz consistently showed evidence of anti-convulsive properties in their iEEG-based seizure profiles when compared to sham rTMS treatment. In contrast, data from 10 Hz rTMS suggested facilitative characteristics. Our results showed that 5 Hz rTMS consistently outperformed 1 Hz rTMS in seizure suppression. This re-emphasizes the importance in accurately characterizing TMS effects on seizure suppression due to the heterogeneous nature of seizures. Thus, finely tuned TMS treatment has great potential to become a powerful asset in combating epilepsy.

Mitochondrial STAT3 is negatively regulated by SOCS3 and upregulated after spinal cord injury

Suppressor of cytokine signaling-3 (SOCS3) expression is induced by the Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) signaling pathway. SOCS3 then acts as a feedback inhibitor of JAK-STAT signaling. Previous studies have shown that knocking down SOCS3 in spinal cord neurons with Lentiviral delivery of SOCS3-targeting shRNA (shSOCS3) increased spinal cord injury (SCI)-induced tyrosine phosphorylation of STAT3 (P-STAT3 Tyr), which in part contributed to decreased neuronal death and demyelination as well as enhanced dendritic regeneration and protection of neuronal morphology after SCI. However, the role of serine phosphorylation of STAT3 (P-STAT3 Ser) is in large part undetermined. Our purposes of this study were to evaluate the expression patterns of P-STAT3 Ser and to explore the possible role of SOCS3 in the regulation of P-STAT3 Ser expression. Immunoblot analyses demonstrated that Oncostatin M (OSM), a member of the interleukin-6 (IL-6) cytokine family, induced both P-STAT3 Tyr and P-STAT3 Ser in SH-SY5Y cells. Subcellular fractionation further revealed that P-STAT3 Ser was localized in mitochondria. Overexpression of SOCS3 with a Lentivirus-mediated approach in SH-SY5Y cells inhibited OSM-induced P-STAT3 Ser in both cytosol and mitochondria fractions. In contrast, OSM-induced P-STAT3 Ser was further upregulated in both cytosol and mitochondria when SOCS3 was knocked down by Lentivirus-delivered shSOCS3. Using a rat T8 spinal cord complete transection model, we found that SCI induced upregulation of P-STAT3 Ser in the mitochondria of macrophages/microglia and neurons both rostral and caudal to the injury site of spinal cord. Collectively, these results suggest that SOCS3 regulation of STAT3 signaling plays critical roles in stress conditions.

Bladder dysfunction changes from underactive to overactive after experimental traumatic brain injury.

Although bladder dysfunction is common after traumatic brain injury (TBI), few studies have investigated resultant bladder changes and the detailed relationship between TBI and bladder dysfunction. The goal of this study was to characterize the effects of TBI on bladder function in an animal model. Fluid-percussion injury was used to create an animal model with moderate TBI. Female Sprague-Dawley rats underwent TBI, sham TBI or were not manipulated (naïve). All rats underwent filling cystometry while bladder pressure and external urethral sphincter electromyograms were simultaneously recorded 1 day, 1 week, 2 weeks, and 1 month after injury. One day after injury, 70% of the animals in the TBI group and 29% of the animals in the sham TBI group showed no bursting activity during urination. Compared to naïve rats, bladder function was mainly altered 1 day and 1 week after sham TBI, suggesting the craniotomy procedure affected bladder function mostly in a temporary manner. Compared to either naïve or sham TBI, bladder weight was significantly increased 1 month after TBI and collagen in the bladder wall was increased. Bladder function in the TBI group went from atonic 1 day post-TBI to overactive 1 month post-TBI, suggesting that TBI significantly affected bladder function.

Neuron-Specific HuR-Deficient Mice Spontaneously Develop Motor Neuron Disease

Human Ag R (HuR) is an RNA binding protein in the ELAVL protein family. To study the neuron-specific function of HuR, we generated inducible, neuron-specific HuR-deficient mice of both sexes. After tamoxifen-induced deletion of HuR, these mice developed a phenotype consisting of poor balance, decreased movement, and decreased strength. They performed significantly worse on the rotarod test compared with littermate control mice, indicating coordination deficiency. Using the grip-strength test, it was also determined that the forelimbs of neuron-specific HuR-deficient mice were much weaker than littermate control mice. Immunostaining of the brain and cervical spinal cord showed that HuR-deficient neurons had increased levels of cleaved caspase-3, a hallmark of cell apoptosis. Caspase-3 cleavage was especially strong in pyramidal neurons and α motor neurons of HuR-deficient mice. Genome-wide microarray and real-time PCR analysis further indicated that HuR deficiency in neurons resulted in altered expression of genes in the brain involved in cell growth, including trichoplein keratin filament–binding protein, Cdkn2c, G-protein signaling modulator 2, immediate early response 2, superoxide dismutase 1, and Bcl2. The additional enriched Gene Ontology terms in the brain tissues of neuron-specific HuR-deficient mice were largely related to inflammation, including IFN-induced genes and complement components. Importantly, some of these HuR-regulated genes were also significantly altered in the brain and spinal cord of patients with amyotrophic lateral sclerosis. Additionally, neuronal HuR deficiency resulted in the redistribution of TDP43 to cytosolic granules, which has been linked to motor neuron disease. Taken together, we propose that this neuron-specific HuR-deficient mouse strain can potentially be used as a motor neuron disease model.