Netosis and Inflammasomes in Large Vessel Occlusion Thrombi.

The inflammatory response appears to play a critical role in clotting in which neutrophil extracellular traps (NETs) are the major drivers of thrombosis in acute ischemic stroke (AIS). The inflammasome is an innate immune complex involved in the activation of interleukin (IL)-18 and IL-1ß through caspase-1, but whether the inflammasome plays a role in NETosis in AIS remains poorly understood. Here we assessed the levels of inflammasome signaling proteins in NETs and their association with clinical and procedural outcomes of mechanical thrombectomy for AIS. Electron microscopy and immunofluorescence indicate the presence of NETs in thrombi of patients with AIS. Moreover, the inflammasome signaling proteins caspase-1 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) were also present in clots associated with the marker of NETosis citrullinated histone 3H (CitH3). Analysis of protein levels by a simple plex assay show that caspase-1, ASC and interleukin (IL)-1ß were significantly elevated in clots when compared to plasma of AIS patients and healthy controls, while IL-18 levels were lower. Moreover, multivariate analyses show that IL-1ß levels in clots contribute to the number of passes to achieve complete recanalization, and that ASC, caspase-1 and IL-18 are significant contributors to time to recanalization. Thus, inflammasome proteins are elevated in NETs present in thrombi of patients with AIS that contribute to poor outcomes following stroke.

Preservation, Sectioning, and Staining of Schwann Cell Cultures for Transmission Electron Microscopy Analysis.

The transmission electron microscope (TEM) enables a unique and valuable examination of cellular and extracellular elements in tissue in situ, in cultured cells, or in pellets derived from suspensions of cells or other materials such as nanoparticles. Here we focus on the preparation of cultured Schwann cells or Schwann cell-containing dorsal root ganglion cultures. To gain as life-like as possible views of the cellular details, it is imperative to achieve excellent preservation of the cellular structure. The steps in the preparation of cultures described in this chapter represent the results of many years of accumulated TEM images to find the best methods of preservation for Schwann cells, myelin, and basal lamina components. All the materials required are listed. The methods for fixing, dehydrating, and embedding a culture are described. Choosing an area in the culture to view, scoring it, cutting it out of the resin-embedded culture, mounting it appropriately for enface or cross-sectioning, and performing the semi-thin and thin sectioning are detailed. Explaining the way in which the sections are then stained for TEM completes the Methods section. Preservation of cultured Schwann cells and their myelin sheaths can be outstanding due to the direct and rapid but careful addition of the fixative solution to the culture dish.

Loss of central inhibition: implications for behavioral hypersensitivity after contusive spinal cord injury in rats.

Behavioral hypersensitivity is common following spinal cord injury (SCI), producing significant discomfort and often developing into chronic pain syndromes. While the mechanisms underlying the development of behavioral hypersensitivity after SCI are poorly understood, previous studies of SCI contusion have shown an increase in amino acids, namely, aspartate and glutamate, along with a decrease in GABA and glycine, particularly below the injury. The current study sought to identify alterations in key enzymes and receptors involved in mediating central inhibition via GABA and glycine after a clinically-relevant contusion SCI model. Following thoracic (T8) 25.0 mm NYU contusion SCI in rodents, significant and persistent behavioral hypersensitivity developed as evidenced by cutaneous allodynia and thermal hyperalgesia. Biochemical analyses confirmed upregulation of glutamate receptor GluR3 with downregulation of the GABA synthesizing enzyme (GAD65/67) and the glycine receptor α3 (GLRA3), notably below the injury. Combined, these changes result in the disinhibition of excitatory impulses and contribute to behavioral hyperexcitability. This study demonstrates a loss of central inhibition and the development of behavioral hypersensitivity in a contusive SCI paradigm. Future use of this model will permit the evaluation of different antinociceptive strategies and help in the elucidation of new targets for the treatment of neuropathic pain.

Limitations of nerve repair of segmental defects using acellular conduits.

The authors present the case of a 20-year-old man who, 3 months after his initial injury, underwent repair of a 1.7-cm defect of the ulnar nerve at the wrist; repair was performed with an acellular nerve allograft. Given the absence of clinical or electrophysiological recovery at 8 months postrepair, the patient underwent reexploration, excision of the "regenerated cable," and rerepair of the ulnar nerve with sural nerve autografts. Histology of the cable demonstrated minimal axonal regeneration at the midpoint of the repair. At the 6- and 12-month follow-ups of the sural nerve graft repair, clinical and electrophysiological evidence of both sensory and motor reinnervation of the ulnar nerve and associated hand muscles was demonstrated. In this report, the authors describe a single case of failed acellular nerve allograft and correlate the results with basic science and human studies reporting length and diameter limitations in human nerve repair utilizing grafts or conduits devoid of viable Schwann cells.

Transplantation of Schwann cells in a collagen tube for the repair of large, segmental peripheral nerve defects in rats.

OBJECT: Segmental nerve defects pose a daunting clinical challenge, as peripheral nerve injury studies have established that there is a critical nerve gap length for which the distance cannot be successfully bridged with current techniques. Construction of a neural prosthesis filled with Schwann cells (SCs) could provide an alternative treatment to successfully repair these long segmental gaps in the peripheral nervous system. The object of this study was to evaluate the ability of autologous SCs to increase the length at which segmental nerve defects can be bridged using a collagen tube. METHODS: The authors studied the use of absorbable collagen conduits in combination with autologous SCs (200,000 cells/µl) to promote axonal growth across a critical size defect (13 mm) in the sciatic nerve of male Fischer rats. Control groups were treated with serum only-filled conduits of reversed sciatic nerve autografts. Animals were assessed for survival of the transplanted SCs as well as the quantity of myelinated axons in the proximal, middle, and distal portions of the channel. RESULTS: Schwann cell survival was confirmed at 4 and 16 weeks postsurgery by the presence of prelabeled green fluorescent protein-positive SCs within the regenerated cable. The addition of SCs to the nerve guide significantly enhanced the regeneration of myelinated axons from the nerve stump into the proximal (p < 0.001) and middle points (p < 0.01) of the tube at 4 weeks. The regeneration of myelinated axons at 16 weeks was significantly enhanced throughout the entire length of the nerve guide (p < 0.001) as compared with their number in a serum-only filled tube and was similar in number compared with the reversed autograft. Autotomy scores were significantly lower in the animals whose sciatic nerve was repaired with a collagen conduit either without (p < 0.01) or with SCs (p < 0.001) when compared with a reversed autograft. CONCLUSIONS: The technique of adding SCs to a guidance channel significantly enhanced the gap distance that can be repaired after peripheral nerve injury with long segmental defects and holds promise in humans. Most importantly, this study represents some of the first essential steps in bringing autologous SC-based therapies to the domain of peripheral nerve injuries with long segmental defects.

Motoneuron replacement for reinnervation of skeletal muscle in adult rats.

Reinnervation is needed to rescue muscle when motoneurons die in disease or injury. Embryonic ventral spinal cord cells transplanted into peripheral nerve reinnervate muscle and reduce atrophy, but low motoneuron survival may limit motor unit formation. We tested whether transplantation of a purified population of embryonic motoneurons into peripheral nerve (mean ± SE, 146,458 ± 4,011 motoneurons) resulted in more motor units and reinnervation than transplantation of a mixed population of ventral spinal cord cells (72,075 ± 12,329 motoneurons). Ten weeks after either kind of transplant, similar numbers of neurons expressed choline acetyl transferase and/or Islet-1. Motoneuron numbers always exceeded the reinnervated motor unit count. Most motor end plate were simple plaques. Reinnervation significantly reduced muscle fiber atrophy. These data show that the number of transplanted motoneurons and motoneuron survival do not limit muscle reinnervation. Incomplete differentiation of motoneurons in nerve and lack of muscle activity may result in immature neuromuscular junctions that limit reinnervation and function.

Neurotrophic factors improve muscle reinnervation from embryonic neurons.

Motoneurons die in diseases like amyotrophic lateral sclerosis and after spinal cord trauma, inducing muscle denervation. We tested whether transplantation of embryonic cells with neurotrophic factors into peripheral nerve of adult rats improves muscle reinnervation and motor unit function more than cells alone. One week after sciatic nerve section, embryonic ventral spinal cord cells were transplanted into the tibial nerve with or without glial cell line-derived neurotrophic factor, hepatocyte growth factor, and insulin-like growth factor-1. These cells represented the only neuron source for muscle reinnervation. Ten weeks after transplantation, all medial gastrocnemius muscles contracted in response to electrical stimulation of cell transplants with factors. Only 80% of muscles responded with cells alone. Factors and cells resulted in survival of more motoneurons and reinnervation of more muscle fibers for a given axon (motor unit) number. Greater reinnervation from embryonic cells may enhance muscle excitation by patterned electrical stimulation.

Embryonic neurons transplanted into the tibial nerve reinnervate muscle and reduce atrophy but NCAM expression persists.

OBJECTIVE: The aim of this study was to use the glycogen depletion technique to determine whether reinnervated muscle fibers could be distinguished from denervated muscle fibers by their size or by neural cell adhesion molecule (NCAM) expression. METHODS: Medial gastrocnemius muscles of five adult Fischer rats were reinnervated from embryonic neurons transplanted into the distal stump of the tibial nerve. Ten weeks later, the transplants were stimulated repeatedly to deplete reinnervated muscle fibers of glycogen. Areas of reinnervated (glycogen-depleted) muscle fibers were measured and assessed for NCAM expression. The areas of muscle fibers from reinnervated, denervated (n=5) and unoperated control muscles (n=5) were compared. RESULTS: Mean reinnervated muscle fiber area was significantly larger than the mean for denervated fibers (mean +/- SE: 40 +/- 6 and 10 +/- 1% of unoperated control fibers, respectively). NCAM was expressed in 55 +/- 7% of reinnervated fibers (mean +/- SE; range: 42-77%). The mean areas of reinnervated fibers that did or did not express NCAM were similar. NCAM was only expressed in some fibers in completely denervated muscles. DISCUSSION: Our data show that NCAM expression does not differentiate muscle denervation or reinnervation. Quantifying the area of large fibers did distinguish reinnervated muscle fibers from denervated fibers and showed that reinnervation of muscle from neurons placed in peripheral nerve is a strategy to rescue muscle from atrophy.