Functional Gait Assessment Using Manual, Semi-Automated and Deep Learning Approaches Following Standardized Models of Peripheral Nerve Injury in Mice.

Objective: To develop a standardized model of stretch−crush sciatic nerve injury in mice, and to compare outcomes of crush and novel stretch−crush injuries using standard manual gait and sensory assays, and compare them to both semi-automated as well as deep-learning gait analysis methods. Methods: Initial studies in C57/Bl6 mice were used to develop crush and stretch−crush injury models followed by histologic analysis. In total, 12 eight-week-old 129S6/SvEvTac mice were used in a six-week behavioural study. Behavioral assessments using the von Frey monofilament test and gait analysis recorded on a DigiGait platform and analyzed through both Visual Gait Lab (VGL) deep learning and standardized sciatic functional index (SFI) measurements were evaluated weekly. At the termination of the study, neurophysiological nerve conduction velocities were recorded, calf muscle weight ratios measured and histological analyses performed. Results: Histological evidence confirmed more severe histomorphological injury in the stretch−crush injured group compared to the crush-only injured group at one week post-injury. Von Frey monofilament paw withdrawal was significant for both groups at week one compared to baseline (p < 0.05), but not between groups with return to baseline at week five. SFI showed hindered gait at week one and two for both groups, compared to baseline (p < 0.0001), with return to baseline at week five. Hind stance width (HSW) showed similar trends as von Frey monofilament test as well as SFI measurements, yet hind paw angle (HPA) peaked at week two. Nerve conduction velocity (NCV), measured six weeks post-injury, at the termination of the study, did not show any significant difference between the two groups; yet, calf muscle weight measurements were significantly different between the two, with the stretch−crush group demonstrating a lower (poorer) weight ratio relative to uninjured contralateral legs (p < 0.05). Conclusion: Stretch−crush injury achieved a more reproducible and constant injury compared to crush-only injuries, with at least a Sunderland grade 3 injury (perineurial interruption) in histological samples one week post-injury in the former. However, serial behavioral outcomes were comparable between the two crush groups, with similar kinetics of recovery by von Frey testing, SFI and certain VGL parameters, the latter reported for the first time in rodent peripheral nerve injury. Semi-automated and deep learning-based approaches for gait analysis are promising, but require further validation for evaluation in murine hind-limb nerve injuries.

Aging and the immune response in diabetic peripheral neuropathy.

A large proportion of older individuals with diabetes go on to develop diabetic peripheral neuropathy (DPN). DPN is associated with an increase in inflammatory cells within the peripheral nerve, activation of nuclear factor kappa-light-chain-enhancer of activated B cells and receptors for advanced glycation end products/advanced glycation end products pathways, aberrant cytokine expression, oxidative stress, ischemia, as well as pro-inflammatory changes in the bone marrow; all processes that may be exacerbated with age. We review the immunological features of DPN and discuss whether age-related changes in relevant immunological areas may contribute to age being a risk factor for DPN.

The immune response and aging in chronic inflammatory demyelinating polyradiculoneuropathy.

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) consists of various autoimmune subtypes in which the peripheral nervous system (PNS) is attacked. CIDP can follow a relapsing-remitting or progressive course where the resultant demyelination caused by immune cells (e.g., T cells, macrophages) and antibodies can lead to disability in patients. Importantly, the age of CIDP patients has a role in their symptomology and specific variants have been associated with differing ages of onset. Furthermore, older patients have a decreased frequency of functional recovery after CIDP insult. This may be related to perturbations in immune cell populations that could exacerbate the disease with increasing age. In the present review, the immune profile of typical CIDP will be discussed followed by inferences into the potential role of relevant aging immune cell populations. Atypical variants will also be briefly reviewed followed by an examination of the available studies on the immunology underlying them.

Presence and activation of pro-inflammatory macrophages are associated with CRYAB expression in vitro and after peripheral nerve injury.

BACKGROUND: Inflammation constitutes both positive and negative aspects to recovery following peripheral nerve injury. Following damage to the peripheral nervous system (PNS), immune cells such as macrophages play a beneficial role in creating a supportive environment for regrowing axons by phagocytosing myelin and axonal debris. However, a prolonged inflammatory response after peripheral nerve injury has been implicated in the pathogenesis of negative symptoms like neuropathic pain. Therefore, the post-injury inflammation must be carefully controlled to prevent secondary damage while allowing for regeneration. CRYAB (also known as alphaB-crystallin/HSPB5) is a small heat shock protein that has many protective functions including an immunomodulatory role in mouse models of multiple sclerosis, spinal cord injury, and stroke. Because its expression wanes and rebounds in the early and late periods respectively after PNS damage, and CRYAB null mice with sciatic nerve crush injury display symptoms of pain, we investigated whether CRYAB is involved in the immune response following PNS injury. METHODS: Sciatic nerve crush injuries were performed in age-matched Cryab knockout (Cryab-/-) and wildtype (WT) female mice. Nerve segments distal to the injury site were processed by immunohistochemistry for macrophages and myelin while protein lysates of the nerves were analyzed for cytokines and chemokines using Luminex and enzyme-linked immunosorbent assay (ELISA). Peritoneal macrophages from the two genotypes were also cultured and polarized into pro-inflammatory or anti-inflammatory phenotypes where their supernatants were analyzed for cytokines and chemokines by ELISA and protein lysates for macrophage antigen presenting markers using western blotting. RESULTS: We report that (1) more pro-inflammatory CD16/32+ macrophages are present in the nerves of Cryab-/- mice at days 14 and 21 after sciatic nerve crush-injury compared to WT counterparts, and (2) CRYAB has an immunosuppressive effect on cytokine secretion [interleukin (IL)-ß, IL-6, IL-12p40, tumor necrosis factor (TNF)-α] from pro-inflammatory macrophages in vitro. CONCLUSIONS: CRYAB may play a role in curbing the potentially detrimental pro-inflammatory macrophage response during the late stages of peripheral nerve regeneration.

The Neuroimmunology of Guillain-Barré Syndrome and the Potential Role of an Aging Immune System.

Guillain-Barré syndrome (GBS) is a paralyzing autoimmune condition affecting the peripheral nervous system (PNS). Within GBS there are several variants affecting different aspects of the peripheral nerve. In general, there appears to be a role for T cells, macrophages, B cells, and complement in initiating and perpetuating attacks on gangliosides of Schwann cells and axons. Of note, GBS has an increased prevalence and severity with increasing age. In addition, there are alterations in immune cell functioning that may play a role in differences in GBS with age alongside general age-related declines in reparative processes (e.g., delayed de-differentiation of Schwann cells and decline in phagocytic ability of macrophages). The present review will explore the immune response in GBS as well as in animal models of several variants of the disorder. In addition, the potential involvement of an aging immune system in contributing to the increased prevalence and severity of GBS with age will be theorized.