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Passive Clearing and 3D Lightsheet Imaging of the Intact and Injured Spinal Cord in Mice.

ABSTRACT: The spinal cord contains a diverse array of sensory and motor circuits that are essential for normal function. Spinal cord injury (SCI) permanently disrupts neural circuits through initial mechanical damage, as well as a cascade of secondary injury events that further expand the spinal cord lesion, resulting in permanent paralysis. Tissue clearing and 3D imaging have recently emerged as promising techniques to improve our understanding of the complex neural circuitry of the spinal cord and the changes that result from damage due to SCI. However, the application of this technology for studying the intact and injured spinal cord remains limited. Here, we optimized the passive CLARITY technique (PACT) to obtain gentle and efficient clearing of the murine spinal cord without the need for specialized equipment. We demonstrate that PACT clearing enables 3D imaging of multiple fluorescent labels in the spinal cord to assess molecularly defined neuronal populations, acute inflammation, long-term tissue damage, and cell transplantation. Collectively, these procedures provide a framework for expanding the utility of tissue clearing to enhance the study of spinal cord neural circuits, as well as cellular- and tissue-level changes that occur following SCI.

SUBMITTER: McCreedy DA

PROVIDER: S-EPMC8366232 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Posterior root-muscle (PRM) reflexes are short-latency spinal reflexes evoked by epidural or transcutaneous spinal cord stimulation (SCS) in clinical and physiological studies. PRM reflexes share key physiological characteristics with the H reflex elicited by electrical stimulation of large-diameter muscle spindle afferents in the tibial nerve. Here, we compared the H reflex and the PRM reflex of soleus in response to transcutaneous stimulation by studying their recovery cycles in ten neurologically intact volunteers and ten individuals with traumatic, chronic spinal cord injury (SCI). The recovery cycles of the reflexes, i.e., the time course of their excitability changes, were assessed by paired pulses with conditioning-test intervals of 20-5000 ms. Between the subject groups, no statistical difference was found for the recovery cycles of the H reflexes, yet those of the PRM reflexes differed significantly, with a striking suppression in the intact group. When comparing the reflex types, they did not differ in the SCI group, while the PRM reflexes were more strongly depressed in the intact group for durations characteristic for presynaptic inhibition. These differences may arise from the concomitant stimulation of several posterior roots containing afferent fibers of various lower extremity nerves by transcutaneous SCS, producing multi-source heteronymous presynaptic inhibition, and the collective dysfunction of inhibitory mechanisms after SCI contributing to spasticity. PRM-reflex recovery cycles additionally obtained for bilateral rectus femoris, biceps femoris, tibialis anterior, and soleus all demonstrated a stronger suppression in the intact group. Within both subject groups, the thigh muscles showed a stronger recovery than the lower leg muscles, which may reflect a characteristic difference in motor control of diverse muscles. Based on the substantial difference between intact and SCI individuals, PRM-reflex depression tested with paired pulses could become a sensitive measure for spasticity and motor recovery.

Project description:Promoting the combination of robust regeneration of damaged axons and synaptic reconnection of these growing axon populations with appropriate neuronal targets represents a major therapeutic goal following spinal cord injury (SCI). A key impediment to achieving this important aim includes an intrinsic inability of neurons to extend axons in adult CNS, particularly in the context of the chronically-injured spinal cord. We tested whether an inhibitory peptide directed against phosphatase and tensin homolog (PTEN: a central inhibitor of neuron-intrinsic axon growth potential) could restore inspiratory diaphragm function by reconnecting critical respiratory neural circuitry in a rat model of chronic cervical level 2 (C2) hemisection SCI. We found that systemic delivery of PTEN antagonist peptide 4 (PAP4) starting at 8 weeks after C2 hemisection promoted substantial, long-distance regeneration of injured bulbospinal rostral Ventral Respiratory Group (rVRG) axons into and through the lesion and back toward phrenic motor neurons (PhMNs) located in intact caudal C3-C5 spinal cord. Despite this robust rVRG axon regeneration, PAP4 stimulated only minimal recovery of diaphragm function. Furthermore, re-lesion through the hemisection site completely removed PAP4-induced functional improvement, demonstrating that axon regeneration through the lesion was responsible for this partial functional recovery. Interestingly, there was minimal formation of putative excitatory monosynaptic connections between regrowing rVRG axons and PhMN targets, suggesting that (1) limited rVRG-PhMN synaptic reconnectivity was responsible at least in part for the lack of a significant functional effect, (2) chronically-injured spinal cord presents an obstacle to achieving synaptogenesis between regenerating axons and post-synaptic targets, and (3) addressing this challenge is a potentially-powerful strategy to enhance therapeutic efficacy in the chronic SCI setting. In conclusion, our study demonstrates a non-invasive and transient pharmacological approach in chronic SCI to repair the critically-important neural circuitry controlling diaphragmatic respiratory function, but also sheds light on obstacles to circuit plasticity presented by the chronically-injured spinal cord.

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Passive Clearing and 3D Lightsheet Imaging of the Intact and Injured Spinal Cord in Mice.

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