The influence of ApoE4 on the clinical outcomes and pathophysiology of degenerative cervical myelopathy.

Degenerative cervical myelopathy (DCM) is the most common cause of nontraumatic spinal cord injury in adults worldwide. Surgical decompression is generally effective in improving neurological outcomes and halting progression of myelopathic deterioration. However, a subset of patients experience suboptimal neurological outcomes. Given the emerging evidence that apolipoprotein E4 (ApoE4) allelic status influences neurodegenerative conditions, we examined whether the presence of the ApoE4 allele may account for the clinical heterogeneity of treatment outcomes in patients with DCM. Our results demonstrate that human ApoE4+ DCM patients have a significantly lower extent of improvement after decompression surgery. Functional analysis of our DCM mouse model in targeted-replacement mice expressing human ApoE4 revealed delayed gait recovery, forelimb grip strength, and hind limb mechanical sensitivity after decompression surgery, compared with their ApoE3 counterparts. This was accompanied by an exacerbated proinflammatory response resulting in higher concentrations of TNF-α, IL-6, CCL3, and CXCL9. At the site of injury, there was a significant decrease in gray matter area, an increase in the activation of microglia/macrophages, and increased astrogliosis after decompression surgery in the ApoE4 mice. Our study is the first to our knowledge to investigate the pathophysiological underpinnings of ApoE4 in DCM, which suggests a possible personalized medicine approach for the treatment of DCM in ApoE4 carriers.

Mir21 modulates inflammation and sensorimotor deficits in cervical myelopathy: data from humans and animal models.

Degenerative cervical myelopathy is a common condition resulting from chronic compression of the spinal cord by degenerating structures of the spine. Degenerative cervical myelopathy present a wide range of outcomes, and the biological factors underlying this variability are poorly understood. Previous studies have found elevated MIR21-5p in the sub-acute and chronic neuroinflammatory environment after spinal cord injury. As chronic spinal cord neuroinflammation is a major feature of degenerative cervical myelopathy, we hypothesized that MIR21-5p may be particularly relevant to disease pathobiology, and could serve as a potential biomarker. A prospective cohort study of 69 human degenerative cervical myelopathy patients (36 male:33 female) between the ages of 30 and 78 years was performed to identify the relationship between MIR21-5p expression, symptom severity and treatment outcomes. Results from this study identified a positive correlation between elevated plasma MIR21-5p expression, initial symptom severity and poor treatment outcomes. Subsequent validation of these relationships using a mouse model of degenerative cervical myelopathy identified a similar elevation of MIR21-5p expression at 6 and 12 weeks after onset, corresponding to moderate to severe neurological deficits. To further determine how MIR21-5p affects cervical myelopathy pathobiology, this mouse model was applied to a Mir21 knockout mouse line. Deletion of the Mir21 gene preserved locomotor function on rotarod and forced swim tests, but also resulted in increased nociception based on tail flick, Von Frey filament and electrophysiological testing. Critically, Mir21 knockout mice also had reduced spinal cord inflammation, demonstrated by the reduction of Iba1+ microglia by ∼50% relative to wild-type controls. In vitro experiments using primary microglial cultures confirmed that MIR21-5p expression was greatly increased after exposure to lipopolysaccharide (pro-inflammatory), Il4 (anti-inflammatory) and hypoxia. Mir21 knockout did not appear to alter the ability of microglia to respond to these stimuli, as expression of key pro- and anti-inflammatory response genes was not significantly altered. However, target prediction algorithms identified the IL6/STAT3 pathway as a potential downstream target of MIR21-5p, and subsequent in vitro testing found that expression of components of the IL6 receptor complex, Il6ra and Il6st, were significantly higher in Mir21 knockout microglia. In aggregate, these data show that Mir21 plays a role in the progression of motor deficits and neuroinflammatory modulation in degenerative cervical myelopathy. Given this role in neuroinflammation, and its association with poor patient outcomes, MIR21-5p represents a potential therapeutic target and a new marker for prognostication.

Changes in Sensorimotor Connectivity to dI3 Interneurons in Relation to the Postnatal Maturation of Grasping.

Primitive reflexes are evident shortly after birth. Many of these reflexes disappear during postnatal development as part of the maturation of motor control. This study investigates the changes of connectivity related to sensory integration by spinal dI3 interneurons during the time in which the palmar grasp reflex gradually disappears in postnatal mice pups. Our results reveal an increase in GAD65/67-labeled terminals to perisomatic Vglut1-labeled sensory inputs contacting cervical and lumbar dI3 interneurons between postnatal day 3 and day 25. In contrast, there were no changes in the number of perisomatic Vglut1-labeled sensory inputs to lumbar and cervical dI3 interneurons other than a decrease between postnatal day 15 and day 25. Changes in postsynaptic GAD65/67-labeled inputs to dI3 interneurons were inconsistent with a role in the sustained loss of the grasp reflex. These results suggest a possible link between the maturation of hand grasp during postnatal development and increased presynaptic inhibition of sensory inputs to dI3 interneurons.

Sensory cortical control of movement.

Walking in our complex environment requires continual higher order integrated spatiotemporal information. This information is processed in the somatosensory cortex, and it has long been presumed that it influences movement via descending tracts originating from the motor cortex. Here we show that neuronal activity in the primary somatosensory cortex tightly correlates with the onset and speed of locomotion in freely moving mice. Using optogenetics and pharmacogenetics in combination with in vivo and in vitro electrophysiology, we provide evidence for a direct corticospinal pathway from the primary somatosensory cortex that synapses with cervical excitatory neurons and modulates the lumbar locomotor network independently of the motor cortex and other supraspinal locomotor centers. Stimulation of this pathway enhances speed of locomotion, while inhibition decreases locomotor speed and ultimately terminates stepping. Our findings reveal a novel pathway for neural control of movement whereby the somatosensory cortex directly influences motor behavior, possibly in response to environmental cues.

Propriospinal Neurons: Essential Elements of Locomotor Control in the Intact and Possibly the Injured Spinal Cord.

Propriospinal interneurons (INs) communicate information over short and long distances within the spinal cord. They act to coordinate different parts of the body by linking motor circuits that control muscles across the forelimbs, trunk, and hindlimbs. Their role in coordinating locomotor circuits near and far may be invaluable to the recovery of locomotor function lost due to injury to the spinal cord where the flow of motor commands from the brain and brainstem to spinal motor circuits is disrupted. The formation and activation of circuits established by spared propriospinal INs may promote the re-emergence of locomotion. In light of progress made in animal models of spinal cord injury (SCI) and in human patients, we discuss the role of propriospinal INs in the intact spinal cord and describe recent studies investigating the assembly and/or activation of propriospinal circuits to promote recovery of locomotion following SCI.

Cervical excitatory neurons sustain breathing after spinal cord injury.

Dysfunctional breathing is the main cause of morbidity and mortality after traumatic injury of the cervical spinal cord1,2 and often necessitates assisted ventilation, thus stressing the need to develop strategies to restore breathing. Cervical interneurons that form synapses on phrenic motor neurons, which control the main inspiratory muscle, can modulate phrenic motor output and diaphragmatic function3-5. Here, using a combination of pharmacogenetics and respiratory physiology assays in different models of spinal cord injury, we show that mid-cervical excitatory interneurons are essential for the maintenance of breathing in mice with non-traumatic cervical spinal cord injury, and are also crucial for promoting respiratory recovery after traumatic spinal cord injury. Although these interneurons are not necessary for breathing under normal conditions, their stimulation in non-injured animals enhances inspiratory amplitude. Immediately after spinal cord injury, pharmacogenetic stimulation of cervical excitatory interneurons restores respiratory motor function. Overall, our results demonstrate a strategy to restore breathing after central nervous system trauma by targeting a neuronal subpopulation.

Delayed decompression exacerbates ischemia-reperfusion injury in cervical compressive myelopathy.

Degenerative cervical myelopathy (DCM) is the most common progressive nontraumatic spinal cord injury. The most common recommended treatment is surgical decompression, although the optimal timing of intervention is an area of ongoing debate. The primary objective of this study was to assess whether a delay in decompression could influence the extent of ischemia-reperfusion injury and alter the trajectory of outcome in DCM. Using a DCM mouse model, we show that decompression acutely led to a 1.5- to 2-fold increase in levels of inflammatory cytokines within the spinal cord. Delayed decompression was associated with exacerbated reperfusion injury, astrogliosis, and poorer neurological recovery. Additionally, delayed decompression was associated with prolonged elevation of inflammatory cytokines and an exacerbated peripheral monocytic inflammatory response (P < 0.01 and 0.001). In contrast, early decompression led to resolution of reperfusion-mediated inflammation, neurological improvement, and reduced hyperalgesia. Similar findings were observed in subjects from the CSM AOSpine North America and International studies, where delayed decompressive surgery resulted in poorer neurological improvement compared with patients with an earlier intervention. Our data demonstrate that delayed surgical decompression for DCM exacerbates reperfusion injury and is associated with ongoing enhanced levels of cytokine expression, microglia activation, and astrogliosis, and paralleled with poorer neurological recovery.

Generating level-dependent models of cervical and thoracic spinal cord injury: Exploring the interplay of neuroanatomy, physiology, and function.

The majority of spinal cord injuries (SCI) occur at the cervical level, which results in significant impairment. Neurologic level and severity of injury are primary endpoints in clinical trials; however, how level-specific damages relate to behavioural performance in cervical injury is incompletely understood. We hypothesized that ascending level of injury leads to worsening forelimb performance, and correlates with loss of neural tissue and muscle-specific neuron pools. A direct comparison of multiple models was made with injury realized at the C5, C6, C7 and T7 vertebral levels using clip compression with sham-operated controls. Animals were assessed for 10weeks post-injury with numerous (40) outcome measures, including: classic behavioural tests, CatWalk, non-invasive MRI, electrophysiology, histologic lesion morphometry, neuron counts, and motor compartment quantification, and multivariate statistics on the total dataset. Histologic staining and T1-weighted MR imaging revealed similar structural changes and distinct tissue loss with cystic cavitation across all injuries. Forelimb tests, including grip strength, F-WARP motor scale, Inclined Plane, and forelimb ladder walk, exhibited stratification between all groups and marked impairment with C5 and C6 injuries. Classic hindlimb tests including BBB, hindlimb ladder walk, bladder recovery, and mortality were not different between cervical and thoracic injuries. CatWalk multivariate gait analysis showed reciprocal and progressive changes forelimb and hindlimb function with ascending level of injury. Electrophysiology revealed poor forelimb axonal conduction in cervical C5 and C6 groups alone. The cervical enlargement (C5-T2) showed progressive ventral horn atrophy and loss of specific motor neuron populations with ascending injury. Multivariate statistics revealed a robust dataset, rank-order contribution of outcomes, and allowed prediction of injury level with single-level discrimination using forelimb performance and neuron counts. Level-dependent models were generated using clip-compression SCI, with marked and reliable differences in forelimb performance and specific neuron pool loss.

Riluzole blocks perioperative ischemia-reperfusion injury and enhances postdecompression outcomes in cervical spondylotic myelopathy.

Although surgical decompression is considered the gold standard treatment for cervical spondylotic myelopathy (CSM), a proportion of cases show postoperative decline or continue to exhibit substantial neurological dysfunction. To investigate this further, we first examined data from the prospective multicenter AOSpine North America CSM study, finding that 9.3% of patients exhibited postoperative functional decline (ΔmJOA, ≤-1) and that 44% of patients were left with substantial neurological impairment 6 months postoperatively. Notably, 4% of patients experienced perioperative neurological complications within 20 days after surgery in otherwise uneventful surgeries. To shed light on the mechanisms underlying this phenomenon and to test a combination therapeutic strategy for CSM, we performed surgical decompression in a rat model of CSM, randomizing some animals to also receive the U.S. Food and Drug Administration-approved drug riluzole. Spinal cord blood flow measurements increased after decompression surgery in rats. CSM rats showed a transient postoperative neurological decline akin to that seen in some CSM patients, suggesting that ischemia-reperfusion injury may occur after decompression surgery. Riluzole treatment attenuated oxidative DNA damage in the spinal cord and postoperative decline after decompression surgery. Mechanistic in vitro studies also demonstrated that riluzole preserved mitochondrial function and reduced oxidative damage in neurons. Rats receiving combined decompression surgery and riluzole treatment displayed long-term improvements in forelimb function associated with preservation of cervical motor neurons and corticospinal tracts compared to rats treated with decompression surgery alone.

Vision deficits precede structural losses in a mouse model of mitochondrial dysfunction and progressive retinal degeneration.

Current animal models of retinal disease often involve the rapid development of a retinal disease phenotype; however, this is at odds with age-related diseases that take many years to manifest clinical symptoms. The present study was performed to examine an apoptosis-inducing factor (Aif)-deficient model, the harlequin carrier mouse (X(hq)X), and determine how mitochondrial dysfunction and subsequent accelerated aging affect the function and structure of the mouse retina. Vision and eye structure for cohorts of 6 X(hq)X and 6 wild type mice at 3, 11, and 15 months of age were studied using in vivo electroretinography (ERG), and optical coherence tomography (OCT). Retinal superoxide levels were determined in situ using dihydroethidium (DHE) histochemistry. Retinal cell counts were quantified post mortem using hematoxylin and eosin (H&E) staining. ERG analysis of X(hq)X retinal function indicated a reduction in b-wave amplitude significant at 3 months of age (p < 0.05), declining further with age. However, retinal neuron counts demonstrated the absence of physical degeneration at 3 and 11 months of age despite significant reduction in ERG b-wave amplitude. Superoxide anion levels were elevated in the ganglion cell, inner nuclear and outer nuclear layers of the retina (p < 0.01, p < 0.01, and p < 0.001, respectively) of 11-month-old X(hq)X mice in comparison to wild type, preceding the structural losses observed at 15 mos. Early onset of retinal function deficits occurred independently of neuron loss. Changes in neurotransmitter localization in the stressed retina may account for the early and significant reduction in retinal function. This remodeling of retinal neurochemistry in response to stress may be a relevant mechanism in the progression of normal retinal aging and early stages of some retinal degenerative diseases.

Ocular pathology relevant to glaucoma in a Gja1(Jrt/+) mouse model of human oculodentodigital dysplasia.

PURPOSE: Oculodentodigital dysplasia (ODDD) is a human disorder caused by mutations in the gap junction alpha 1 (GJA1) gene encoding the connexin43 (Cx43) gap junction protein. Causal links between GJA1 mutations and glaucoma are not understood. The purpose in this study was to examine the ocular phenotype for Gja1(Jrt/+) mice harboring a Cx43 G60S mutation. METHODS; In young Gja1(Jrt/+) mice, Cx43 abundance was assessed with a Western blot, and Cx43 localization was visualized using immunohistochemistry and confocal microscopy. Intraocular pressure (IOP) was measured by rebound tonometry, and eye anatomy was imaged using ocular coherence tomography (OCT). Hematoxylin and eosin (H&E)-stained eye sections were examined for ocular histopathology related to the development of glaucoma. RESULTS: Decreased Cx43 protein levels were evident in whole eyes from Gja1(Jrt/+) mice compared with those of wild-type mice at postnatal day 1 (P = 0.005). Cx43 immunofluorescence in ciliary bodies of Gja1(Jrt/+) mice was diffuse and intracellular, unlike the gap junction plaques prevalent in wild-type mice. IOP in Gja1(Jrt/+) mice changed during postnatal development, with significantly lower IOP at 21 weeks of age in comparison to the IOP of wild-type eyes. Microphthalmia, enophthalmia, anterior angle closure, and reduced pupil diameter were observed in Gja1(Jrt/+) mice at all ages examined. Ocular histology showed prominent separations between the pigmented and nonpigmented ciliary epithelium of Gja1(Jrt/+) mice, split irides, and alterations in the number and distribution of nuclei in the retina. CONCLUSIONS: Detailed phenotyping of Gja1(Jrt/+) eyes offers a framework for elucidating human ODDD ocular disease mechanisms and evaluating new treatments designed to protect ocular synaptic network integrity.