Clearing-enabled light sheet microscopy as a novel method for three-dimensional mapping of the sensory innervation of the mouse knee.

A major barrier that hampers our understanding of the precise anatomic distribution of pain sensing nerves in and around the joint is the limited view obtained from traditional two dimensional (D) histological approaches. Therefore, our objective was to develop a workflow that allows examination of the innervation of the intact mouse knee joint in 3D by employing clearing-enabled light sheet microscopy. We first surveyed existing clearing protocols (SUMIC, PEGASOS, and DISCO) to determine their ability to clear the whole mouse knee joint, and discovered that a DISCO protocol provided the most optimal transparency for light sheet microscopy imaging. We then modified the DISCO protocol to enhance binding and penetration of antibodies used for labeling nerves. Using the pan-neuronal PGP9.5 antibody, our protocol allowed 3D visualization of innervation in and around the mouse knee joint. We then implemented the workflow in mice intra-articularly injected with nerve growth factor (NGF) to determine whether changes in the nerve density can be observed. Both 3D and 2D analytical approaches of the light sheet microscopy images demonstrated quantifiable changes in midjoint nerve density following 4 weeks of NGF injection in the medial but not in the lateral joint compartment. We provide, for the first time, a comprehensive workflow that allows detailed and quantifiable examination of mouse knee joint innervation in 3D.

Reduced Capsaicin-Induced Mechanical Allodynia and Neuronal Responses in the DRG in the Presence of Ptpn6 Overexpression.

Transient Receptor Potential Vanilloid 1 (TRPV1) is a nonselective cation channel expressed by pain-sensing neurons and has been an attractive target for the development of drugs to treat pain. Recently, Src homology region 2 domain-containing phosphatase-1 (SHP-1, encoded by Ptpn6) was shown to dephosphorylate TRPV1 in dorsal root ganglia (DRG) neurons, which was linked with alleviating different pain phenotypes. These previous studies were performed in male rodents only and did not directly investigate the role of SHP-1 in TRPV-1 mediated sensitization. Therefore, our goal was to determine the impact of Ptpn6 overexpression on TRPV1-mediated neuronal responses and capsaicin-induced pain behavior in mice of both sexes. Twelve-week-old male and female mice overexpressing Ptpn6 (Shp1-Tg) and their wild type (WT) littermates were used. Ptpn6 overexpression was confirmed in the DRG of Shp1-Tg mice by RNA in situ hybridization and RT-qPCR. Trpv1 and Ptpn6 were found to be co-expressed in DRG sensory neurons in both genotypes. Functionally, this overexpression resulted in lower magnitude intracellular calcium responses to 200 nM capsaicin stimulation in DRG cultures from Shp1-Tg mice compared to WTs. In vivo, we tested the effects of Ptpn6 overexpression on capsaicin-induced pain through a model of capsaicin footpad injection. While capsaicin injection evoked nocifensive behavior (paw licking) and paw swelling in both genotypes and sexes, only WT mice developed mechanical allodynia after capsaicin injection. We observed similar level of TRPV1 protein expression in the DRG of both genotypes, however, a higher amount of tyrosine phosphorylated TRPV1 was detected in WT DRG. These experiments suggest that, while SHP-1 does not mediate the acute swelling and nocifensive behavior induced by capsaicin, it does mediate a protective effect against capsaicin-induced mechanical allodynia in both sexes. The protective effect of SHP-1 might be mediated by TRPV1 dephosphorylation in capsaicin-sensitive sensory neurons of the DRG.

Piezo2 expressing nociceptors mediate mechanical sensitization in experimental osteoarthritis.

Non-opioid targets are needed for addressing osteoarthritis pain, which is mechanical in nature and associated with daily activities such as walking and climbing stairs. Piezo2 has been implicated in the development of mechanical pain, but the mechanisms by which this occurs remain poorly understood, including the role of nociceptors. Here we show that nociceptor-specific Piezo2 conditional knock-out mice were protected from mechanical sensitization associated with inflammatory joint pain in female mice, joint pain associated with osteoarthritis in male mice, as well as both knee swelling and joint pain associated with repeated intra-articular injection of nerve growth factor in male mice. Single cell RNA sequencing of mouse lumbar dorsal root ganglia and in situ hybridization of mouse and human lumbar dorsal root ganglia revealed that a subset of nociceptors co-express Piezo2 and Ntrk1 (the gene that encodes the nerve growth factor receptor TrkA). These results suggest that nerve growth factor-mediated sensitization of joint nociceptors, which is critical for osteoarthritic pain, is also dependent on Piezo2, and targeting Piezo2 may represent a therapeutic option for osteoarthritis pain control.

Exposure to 5 cGy 28Si Particles Induces Long-Term Microglial Activation in the Striatum and Subventricular Zone and Concomitant Neurogenic Suppression.

The proposed mission to Mars will expose astronauts to space radiation that is known to adversely affect cognition and tasks that rely on fine sensorimotor function. Space radiation has also been shown to affect the microglial and neurogenic responses in the central nervous system (CNS). We recently reported that a low dose of 5 cGy 600 MeV/n 28Si results in impaired cognition and skilled motor behavior in adult rats. Since these tasks rely at least in part on the proper functioning of the striatum, we examined striatal microglial cells in these same subjects. Using morphometric analysis, we found that 28Si exposure increased activated microglial cells in the striatum. The majority of these striatal Iba1+ microglia were ED1-, indicating that they were in an alternatively activated state, where microglia do not have phagocytic activity but may be releasing cytokines that could negatively impact neuronal function. In the other areas studied, Iba1+ microglial cells were increased in the subventricular zone (SVZ), but not in the dentate gyrus (DG). Additionally, we examined the relationship between the microglial response and neurogenesis. An analysis of new neurons in the DG revealed an increase in doublecortin-positive (DCX+) hilar ectopic granule cells (hEGC) which correlated with Iba1+ cells, suggesting that microglial cells contributed to this aberrant distribution which may adversely affect hippocampal function. Taken together, these results indicate that a single dose of 28Si radiation results in persistent cellular effects in the CNS that may impact astronauts both in the short and long-term following deep space missions.

Improved Functional Outcome After Peripheral Nerve Stimulation of the Impaired Forelimb Post-stroke.

Lack of blood flow to the brain, i.e., ischemic stroke, results in loss of nerve cells and therefore loss of function in the effected brain regions. There is no effective treatment to improve lost function except restoring blood flow within the first several hours. Rehabilitation strategies are widely used with limited success. The purpose of this study was to examine the effect of electrical stimulation on the impaired upper extremity to improve functional recovery after stroke. We developed a rodent model using an electrode cuff implant onto a single peripheral nerve (median nerve) of the paretic forelimb and applied daily electrical stimulation. The skilled forelimb reaching test was used to evaluate functional outcome after stroke and electrical stimulation. Anterograde axonal tracing from layer V pyramidal neurons with biotinylated dextran amine was done to evaluate the formation of new neuronal connections from the contralesional cortex to the deafferented spinal cord. Rats receiving electrical stimulation on the median nerve showed significant improvement in the skilled forelimb reaching test in comparison with stroke only and stroke with sham stimulation. Rats that received electrical stimulation also exhibited significant improvement in the latency to initiate adhesive removal from the impaired forelimb, indicating better sensory recovery. Furthermore, axonal tracing analysis showed a significant higher midline fiber crossing index in the cervical spinal cord of rats receiving electrical stimulation. Our results indicate that direct peripheral nerve stimulation leads to improved sensorimotor recovery in the stroke-impaired forelimb, and may be a useful approach to improve post-stroke deficits in human patients.

Skilled movement and posture deficits in rat string-pulling behavior following low dose space radiation (28Si) exposure.

Deep space flight missions beyond the Van Allen belt have the potential to expose astronauts to space radiation which may damage the central nervous system and impair function. The proposed mission to Mars will be the longest mission-to-date and identifying mission critical tasks that are sensitive to space radiation is important for developing and evaluating the efficacy of counter measures. Fine motor control has been assessed in humans, rats, and many other species using string-pulling behavior. For example, focal cortical damage has been previously shown to disrupt the topographic (i.e., path circuity) and kinematic (i.e., moment-to-moment speed) organization of rat string-pulling behavior count to compromise task accuracy. In the current study, rats were exposed to a ground-based model of simulated space radiation (5 cGy 28Silicon), and string-pulling behavior was used to assess fine motor control. Irradiated rats initially took longer to pull an unweighted string into a cage, exhibited impaired accuracy in grasping the string, and displayed postural deficits. Once rats were switched to a weighted string, some deficits lessened but postural instability remained. These results demonstrate that a single exposure to a low dose of space radiation disrupts skilled hand movements and posture, suggestive of neural impairment. This work establishes a foundation for future studies to investigate the neural structures and circuits involved in fine motor control and to examine the effectiveness of counter measures to attenuate the effects of space radiation on fine motor control.

Dentate Gyrus Proliferative Responses After Traumatic Brain Injury and Binge Alcohol in Adult Rats.

BACKGROUND: Traumatic brain injury is a significant public health issue that results in serious disability in survivors. Traumatic brain injury patients are often intoxicated with alcohol when admitted to the hospital; however, it is not clear how acute intoxication affects recovery from a traumatic brain injury. Our group has previously shown that binge alcohol prior to traumatic brain injury resulted in long-term impairment in a fine sensorimotor task that was correlated with a decreased proliferative and neuroblast response from the subventricular zone. However, whether binge alcohol prior to traumatic brain injury affects the proliferative response in the hippocampal dentate gyrus is not yet known. METHODS: Male rats underwent binge alcohol (3 g/kg/day) by gastric gavage for 3 days prior to traumatic brain injury. Cell proliferation was labeled by BrdU injections following traumatic brain injury. Stereological quantification and immunofluorescence confocal analysis of BrdU+ cells in the hippocampal dorsal dentate gyrus was performed at 24 hours, 1 week and 6 weeks post traumatic brain injury. RESULTS: We found that either traumatic brain injury alone or binge alcohol alone significantly increased dentate gyrus proliferation at 24 hours and 1 week. However, a combined binge alcohol and traumatic brain injury regimen resulted in decreased dentate gyrus proliferation at 24 hours post-traumatic brain injury. At the 6 week time point, binge alcohol overall reduced the number of BrdU+ cells. Furthermore, more BrdU+ cells were found in the dentate hilar region of alcohol traumatic brain injury compared to vehicle traumatic brain injury groups. The location and double-labeling of these mismigrated BrdU+ cells was consistent with hilar ectopic granule cells. CONCLUSION: The results from this study showed that pre-traumatic brain injury binge alcohol impacts the injury-induced proliferative response in the dentate gyrus in the short-term and may affect the distribution of newly generated cells in the dentate gyrus in the long-term.