Sensorimotor dysfunction in a mild mouse model of cortical contusion injury without significant neuronal loss is associated with increases in inflammatory proteins with innate but not adaptive immune functions.

Traumatic brain injury is a leading cause of mortality and morbidity in the United States. Acute trauma to the brain triggers chronic secondary injury mechanisms that contribute to long-term neurological impairment. We have developed a single, unilateral contusion injury model of sensorimotor dysfunction in adult mice. By targeting a topographically defined neurological circuit with a mild impact, we are able to track sustained behavioral deficits in sensorimotor function in the absence of tissue cavitation or neuronal loss in the contused cortex of these mice. Stereological histopathology and multiplex enzyme-linked immunosorbent assay proteomic screening confirm contusion resulted in chronic gliosis and the robust expression of innate immune cytokines and monocyte attractant chemokines IL-1ß, IL-5, IL-6, TNFα, CXCL1, CXCL2, CXCL10, CCL2, and CCL3 in the contused cortex. In contrast, the expression of neuroinflammatory proteins with adaptive immune functions was not significantly modulated by injury. Our data support widespread activation of innate but not adaptive immune responses, confirming an association between sensorimotor dysfunction with innate immune activation in the absence of tissue or neuronal loss in our mice.

Host sex and transplanted human induced pluripotent stem cell phenotype interact to influence sensorimotor recovery in a mouse model of cortical contusion injury.

Traumatic brain injury (TBI) is a substantial cause of disability and death worldwide. Primary head trauma triggers chronic secondary injury mechanisms in the brain that are a focus of therapeutic efforts to treat TBI. Currently, there is no successful clinical strategy to repair brain injury. Cell transplantation therapies have demonstrated promise in attenuating secondary injury mechanisms of neuronal death and dysfunction in animal models of brain injury. In this study, we used a unilateral cortical contusion injury (CCI) model of sensorimotor brain injury to examine the effects of human induced pluripotent stem cell (hiPSC) transplantation on pathology in male and female adult mice. We determined transplanted hiPSC-derived neural stem cells (NSCs) and neuroblasts but not astrocytes best tolerate the injured host environment. Surviving NSC and neuroblast cells were clustered at the site of injection within the deep layers of the cortex and underlying corpus callosum. Cell grafts extended neuritic processes that crossed the midline into the contralateral corpus callosum or continued laterally within the external capsule to enter the ipsilateral entorhinal cortex. To determine the effect of transplantation on neuropathology, we performed sensorimotor behavior testing and stereological estimation of host neurons, astrocytes, and microglia within the contused cortex. These measures did not reveal a consistent effect of transplantation on recovery post-injury. Rather the positive and negative effects of cell transplantation were dependent on the host sex, highlighting the importance of developing patient-specific approaches to treat TBI. Our study underscores the complex interactions of sex, neuroimmune responses and cell therapy in a common experimental model of TBI.

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

Anesthetics disrupt growth cone guidance cue sensing through actions on the GABAA α2 receptor mediated by the immature chloride gradient.

BACKGROUND: General anesthetics (GAs) may exert harmful effects on the developing brain by disrupting neuronal circuit formation. Anesthetics that act on γ-aminobutyric acid (GABA) receptors can interfere with axonal growth cone guidance, a critical process in the assembly of neuronal circuitry. Here we investigate the mechanism by which isoflurane prevents sensing of the repulsive guidance cue, Semaphorin 3A (Sema3A). METHODS: Growth cone sensing was assayed by measuring growth cone collapse in dissociated neocortical cultures exposed to recombinant Sema3A in the presence or absence of isoflurane and/or a panel of reagents with specific actions on components of the GABA receptor and chloride ion systems. RESULTS: Isoflurane exposure prevents Sema3A induced growth cone collapse. A GABAA α2 specific agonist replicates this effect (36.83 ±â€¯3.417% vs 70.82 ±â€¯2.941%, in the Sema3A induced control group, p < 0.0001), but an α1-specific agonist does not. Both a Na-K-Cl cotransporter 1 antagonism (bumetanide, BUM) and a chloride ionophore (IONO) prevent isoflurane from disrupting growth cone sensing of Sema3A. (65.67 ±â€¯3.775% in Iso + BUM group vs 67.45 ±â€¯3.624% in Sema3A induced control group, 65.34 ±â€¯1.678% in Iso + IONO group vs 68.71 ±â€¯2.071% in Sema3A induced control group, no significant difference) (n = 96 growth cones per group). CONCLUSION: Our data suggest that the effects of isoflurane on growth cone sensing are mediated by the α2 subunit of the GABAA receptor and also that they are dependent on the developmental chloride gradient, in which Cl- exhibits a depolarizing effect. These findings provide a rationale for why immature neurons are particularly susceptible to anesthetic toxicity.

Successful treatment of choreo-athetotic movements in a patient with an EEF1A2 gene variant.

Pathogenic variants in EEF1A2, a gene encoding a eukaryotic translation elongation factor, have been previously reported in pediatric cases of epileptic encephalopathy and intellectual disability. We report a case of a 17-year-old male with a prior history of epilepsy, autism, intellectual disability, and the abrupt onset of choreo-athetotic movements. The patient was diagnosed with an EEF1A2 variant by whole exome sequencing. His movement disorder responded dramatically to treatment with tetrabenazine. To the best of our knowledge, this is the first report of successful treatment of a hyperkinetic movement disorder in the setting of EEF1A2 mutation. A trial with tetrabenazine should be considered in cases with significant choreoathetosis.

Conditional deletion of Neurog1 in the cerebellum of postnatal mice delays inhibitory interneuron maturation.

The transcriptional programs that drive the generation of diverse GABAergic neuron populations from their common progenitor pools in the developing cerebellum remain unclear. Neurog1 is a pro-neural basic helix-loop-helix transcription factor expressed in GABAergic progenitor cells in the ventricular zone (VZ) of embryos and subsequently in the presumptive white matter (pWM) tracts of developing postnatal mice. Genetic inducible fate-mapping labels Purkinje cells and all inhibitory interneuron cell types of the cerebellar cortex. As conventional Neurog1Neo knockout (KO) mice are neonatal lethal, we generated Neurog1loxP mutant mice to examine the effects of conditional Neurog1 deletion on the postnatal development of the cerebellum. Targeted Neurog1 loss-of-function in the developing cerebellum does not result in significant differences in cerebellar morphology or in the number of GABAergic neurons in the cerebellar cortex of mice at postnatal day 21 (P21). To determine the effects of Neurog1 deletion on GABAergic progenitors, we quantified rates of cell proliferation and cell cycle progression or re-entry in embryonic Neurog1Neo and postnatal Neurog1loxP mutants. The data revealed no significant effect of Neurog1 loss-of-function on embryonic day 12.5 (E12.5) VZ progenitors or on P5 and P6 progenitors in the pWM at P7. However, 4-5 day pulse-labeling of P5 and P6 progenitors revealed reductions in inhibitory interneuron dispersal from the pWM to the cerebellar cortex in P10 conditional Neurog1loxP/loxP KO mice. Thus, our conditional Neurog1 KO approach reveals a requirement for Neurog1 activity in inhibitory interneuron cell dispersal from pWM tracts in the developing cerebellum of postnatal mice.

Sensitivity to isoflurane anesthesia increases in autism spectrum disorder Shank3+/∆c mutant mouse model.

Autism is a heterogeneous developmental disorder characterized by impaired social interaction, impaired communication skills, and restricted and repetitive behavior. The abnormal behaviors of these patients can make their anesthetic and perioperative management difficult. Evidence in the literature suggests that some patients with autism or specific autism spectrum disorders (ASD) exhibit altered responses to pain and to anesthesia or sedation. A genetic mouse model of one particular ASD, Phelan McDermid Syndrome, has been developed that has a Shank3 haplotype truncation (Shank3+/Δc). These mice exhibit important characteristics of autism that mimic human autistic behavior. Our study demonstrates that a Shank3+/ΔC mutation in mice is associated with a reduction in both the MAC and RREC50 of isoflurane and down regulation of NR1 in vestibular nuclei and PSD95 in spinal cord. Decreased expression of NR1 and PSD95 in the central nervous system of Shank3+/ΔC mice could help reduce the MAC and RREC50 of isoflurane, which would warrant confirmation in a clinical study. If Shank3 mutations are found to affect anesthetic sensitivity in patients with ASD, better communication and stricter monitoring of anesthetic depth may be necessary.

Neurogenesis and developmental anesthetic neurotoxicity.

The mechanism by which anesthetics might act on the developing brain in order to cause long term deficits remains incompletely understood. The hippocampus has been identified as a structure that is likely to be involved, as rodent models show numerous deficits in behavioral tasks of learning that are hippocampal-dependent. The hippocampus is an unusual structure in that it is the site of large amounts of neurogenesis postnatally, particularly in the first year of life in humans, and these newly generated neurons are critical to the function of this structure. Intriguingly, neurogenesis is a major developmental event that occurs during postulated windows of vulnerability to developmental anesthetic neurotoxicity across the different species in which it has been studied. In this review, we examine the evidence for anesthetic effects on neurogenesis in the early postnatal period and ask whether neurogenesis should be studied further as a putative mechanism of injury. Multiple anesthetics are considered, and both in vivo and in vitro work is presented. While there is abundant evidence that anesthetics act to suppress neurogenesis at several different phases, evidence of a causal link between these effects and any change in learning behavior remains elusive.

Effects of Neonatal Hypoxic-Ischemic Injury and Hypothermic Neuroprotection on Neural Progenitor Cells in the Mouse Hippocampus.

Neonatal hypoxic-ischemic injury (HI) results in widespread cerebral encephalopathy and affects structures that are essential for neurocognitive function, such as the hippocampus. The dentate gyrus contains a reservoir of neural stem and progenitor cells (NSPCs) that are critical for postnatal development and normal adult function of the hippocampus, and may also facilitate the recovery of function after injury. Using a neonatal mouse model of mild-to-moderate HI and immunohistochemical analysis of NSPC development markers, we asked whether these cells are vulnerable to HI and how they respond to both injury and hypothermic therapy. We found that cleaved caspase-3 labeling in the subgranular zone, where NSPCs are located, is increased by more than 30-fold after HI. The population of cells positive for both proliferating cell nuclear antigen and nestin (PCNA+Nes+), which represent primarily actively proliferating NSPCs, are acutely decreased by 68% after HI. The NSPC population expressing NeuroD1, a marker for NSPCs transitioning to become fate-committed neural progenitors, was decreased by 47%. One week after HI, there was a decrease in neuroblasts and immature neurons in the dentate gyrus, as measured by doublecortin (DCX) immunolabeling, and at the same time PCNA+Nes+ cell density was increased by 71%. NSPCs expressing Tbr2, which identifies a highly proliferative intermediate neural progenitor population, increased by 107%. Hypothermia treatment after HI partially rescues both the acute decrease in PCNA+Nes+ cell density at 1 day after injury and the chronic loss of DCX immunoreactivity and reduction in NeuroD1 cell density measured at 1 week after injury. Thus, we conclude that HI causes an acute loss of dentate gyrus NSPCs, and that hypothermia partially protects NSPCs from HI.

Stress induces pain transition by potentiation of AMPA receptor phosphorylation.

Chronic postsurgical pain is a serious issue in clinical practice. After surgery, patients experience ongoing pain or become sensitive to incident, normally nonpainful stimulation. The intensity and duration of postsurgical pain vary. However, it is unclear how the transition from acute to chronic pain occurs. Here we showed that social defeat stress enhanced plantar incision-induced AMPA receptor GluA1 phosphorylation at the Ser831 site in the spinal cord and greatly prolonged plantar incision-induced pain. Interestingly, targeted mutation of the GluA1 phosphorylation site Ser831 significantly inhibited stress-induced prolongation of incisional pain. In addition, stress hormones enhanced GluA1 phosphorylation and AMPA receptor-mediated electrical activity in the spinal cord. Subthreshold stimulation induced spinal long-term potentiation in GluA1 phosphomimetic mutant mice, but not in wild-type mice. Therefore, spinal AMPA receptor phosphorylation contributes to the mechanisms underlying stress-induced pain transition.

Role of neuregulin-1/ErbB signaling in stem cell therapy for spinal cord injury-induced chronic neuropathic pain.

Chronic neuropathic pain is a common and debilitating consequence of spinal cord injury (SCI). In a rat contusion injury model, we observed that chronic neuropathic pain is present on day 7 after SCI and persists for the entire 56-day observation period. However, currently available pain therapies are inadequate for SCI-induced neuropathic pain. In this study, we show that spinal transplantation of mouse embryonic stem cell-derived oligodendrocyte progenitor cells (OPCs) enhances remyelination in the injured spinal cord and reduces SCI-induced chronic neuropathic pain. Moreover, we found that SCI reduces the protein level of neuregulin-1 and ErbB4 in the injured spinal cord and that OPC transplantation enhances the spinal expression of both proteins after SCI. Finally, intrathecal injection of neuregulin-1 small interfering RNA, but not the control nontarget RNA, diminishes OPC transplantation-produced remyelination and reverses the antinociceptive effect of OPC transplantation. Our findings suggest that the transplantation of embryonic stem cell-derived OPCs is an appropriate therapeutic intervention for treatment of SCI-induced chronic neuropathic pain, and that neuregulin-1/ErbB signaling plays an important role in central remyelination under pathological conditions and contributes to the alleviation of such pain.

Predifferentiated GABAergic neural precursor transplants for alleviation of dysesthetic central pain following excitotoxic spinal cord injury.

Intraspinal quisqualic acid (QUIS) injury induce (i) mechanical and thermal hyperalgesia, (ii) progressive self-injurious overgrooming of the affected dermatome. The latter is thought to resemble painful dysesthesia observed in spinal cord injury (SCI) patients. We have reported previously loss of endogenous GABA immunoreactive (IR) cells in the superficial dorsal horn of QUIS rats 2 weeks post injury. Further histological evaluation showed that GABA-, glycine-, and synaptic vesicular transporter VIAAT-IR persisted but were substantially decreased in the injured spinal cord. In this study, partially differentiated GABA-IR embryonic neural precursor cells (NPCs) were transplanted into the spinal cord of QUIS rats to reverse overgrooming by replenishing lost inhibitory circuitry. Rat E14 NPCs were predifferentiated in 0.1 ng/ml FGF-2 for 4 h prior to transplantation. In vitro immunocytochemistry of transplant cohort showed large population of GABA-IR NPCs that double labeled with nestin but few colocalized with NeuN, indicating partial maturation. Two weeks following QUIS lesion at T12-L1, and following the onset of overgrooming, NPCs were transplanted into the QUIS lesion sites; bovine adrenal fibroblast cells were used as control. Overgrooming was reduced in >55.5% of NPC grafted animals, with inverse relationship between the number of surviving GABA-IR cells and the size of overgrooming. Fibroblast-control animals showed a progressive worsening of overgrooming. At 3 weeks post-transplantation, numerous GABA-, nestin-, and GFAP-IR cells were present in the lesion site. Surviving grafted GABA-IR NPCs were NeuN(+) and GFAP(-). These results indicate that partially differentiated NPCs survive and differentiate in vivo into neuronal cells following transplantation into an injured spinal cord. GABA-IR NPC transplants can restore lost dorsal horn inhibitory signaling and are useful in alleviating central pain following SCI.

Combined extrinsic and intrinsic manipulations exert complementary neuronal enrichment in embryonic rat neural precursor cultures: an in vitro and in vivo analysis.

Numerous central nervous system (CNS) disorders share a common pathology in dysregulation of gamma-aminobutyric acid (GABA) inhibitory signaling. Transplantation of GABA-releasing cells at the site of disinhibition holds promise for alleviating disease symptoms with fewer side effects than traditional drug therapies. We manipulated fibroblast growth factor (FGF)-2 deprivation and mammalian achaete-scute homolog (MASH)1 transcription factor levels in an attempt to amplify the default GABAergic neuronal fate in cultured rat embryonic neural precursor cells (NPCs) for use in transplantation studies. Naïve and MASH1 lentivirus-transduced NPCs were maintained in FGF-2 or deprived of FGF-2 for varying lengths of time. Immunostaining and quantitative analysis showed that GABA- and beta-III-tubulin-immunoreactive cells generally decreased through successive passages, suggesting a loss of neurogenic potential in rat neurospheres expanded in vitro. However, FGF-2 deprivation resulted in a small, but significantly increased population of GABAergic cells derived from passaged neurospheres. In contrast to naïve and GFP lentivirus-transduced clones, MASH1 transduction resulted in increased bromodeoxyuridine (BrdU) incorporation and clonal colony size. Western blotting showed that MASH1 overexpression and FGF-2 deprivation additively increased beta-III-tubulin and decreased cyclic nucleotide phosphodiesterase (CNPase) expression, whereas FGF-2 deprivation alone attenuated glial fibrillary acidic protein (GFAP) expression. These results suggest that low FGF-2 signaling and MASH1 activity can operate in concert to enrich NPC cultures for a GABA neuronal phenotype. When transplanted into the adult rat spinal cord, this combination also yielded GABAergic neurons. These findings indicate that, even for successful utilization of the default GABAergic neuronal precursor fate, a combination of both extrinsic and intrinsic manipulations will likely be necessary to realize the full potential of NSC grafts in restoring function.

Sustained analgesic peptide secretion and cell labeling using a novel genetic modification.

Cell-based therapy for neuropathic pain could provide analgesics to local pain modulatory regions in a sustained, renewable fashion. In order to provide enhanced analgesic efficacy, transplantable cells may be engineered to produce complementary or increased levels of analgesic peptides. In addition, genetic labeling of modified cells is desirable for identification and tracking, but it should be retained intracellularly as desired analgesic peptides are secreted. Usually constructs encode proteins destined for either extra- or intracellular compartments, as these pathways do not cross. However, interactions between intracellular destinations provide a window of opportunity to overcome this limitation. In this report, we have explored this approach using a potential supplementary analgesic peptide, [Ser1]-histogranin (SHG), the stable synthetic derivative of a naturally occurring peptide with N-methyl D-aspartate (NMDA) antagonistic properties. A synthetic SHG gene was combined with (i) nerve growth factor-beta (NGF-beta) amino-terminal signal peptide to enable secretion, and (ii) a fluorescent cellular label (mRFP) with intervening cathepsin L cleavage site, and subcloned into a lentiviral vector. In addition, an endoplasmic retention signal, KDEL, was added to enable retrieval of mRFP. Using immunocytochemistry and confocal microscopic profile analysis, cells transduced by such lentiviruses were shown to synthesize a single SHG-mRFP polypeptide that was processed, targeted to expected subcellular destinations in several cell types. Dot blot and Western analysis revealed stable transduction and long-term secretion of SHG from PC12 cells in vitro. Transplantation of such cells provided modest analgesia in a rodent pain model consistent with low levels of SHG peptide in the cerebrospinal fluid (CSF). These results suggest that it is possible to deliver proteins with different final destinations from a single construct, such as pharmacologically active peptide for secretion and intracellular label for identifying transplantable cells.

Prolonged nociceptive responses to hind paw formalin injection in rats with a spinal cord injury.

Unilateral lesioning of the spinal dorsal horn with the excitotoxin quisqualic acid (QUIS) leads to robust degeneration of dorsal horn grey matter, and robust pain-related symptoms, such as cutaneous hypersensitivity, persist long after injury. A possible mechanism that underlies the pain-related symptoms is the disruption of dorsal horn inhibitory neuron function, leading to decreased inhibition of nociceptive neurons. Five percent formalin was injected into the hind paw of rats with either a QUIS lesion or sham lesion. Both QUIS- and sham-lesioned rats displayed bi-phasic hind paw flinches following formalin injection, but a prolonged response was observed in QUIS-lesioned rats. The expression of the immediate-early gene product Fos in the dorsal horn ipsilateral to formalin injection was similar between QUIS- and sham-lesioned rats. In QUIS-lesioned rats, however, there was a marked absence of dorsal horn neurons, particularly GABAergic neurons, compared to sham-lesioned rats. The prolonged nociceptive response observed with a unilateral QUIS lesion may be due to generalized changes in dorsal horn neuron function including a loss of inhibitory neuron function.