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.

Cystatin C Plays a Sex-Dependent Detrimental Role in Experimental Autoimmune Encephalomyelitis.

The cysteine protease inhibitor Cystatin C (CST3) is highly expressed in the brains of multiple sclerosis (MS) patients and C57BL/6J mice with experimental autoimmune encephalomyelitis (EAE; a model of MS), but its roles in the diseases are unknown. Here, we show that CST3 plays a detrimental function in myelin oligodendrocyte glycoprotein 35-55 (MOG35-55)-induced EAE but only in female animals. Female Cst3 null mice display significantly lower clinical signs of disease compared to wild-type (WT) littermates. This difference is associated with reduced interleukin-6 production and lower expression of key proteins (CD80, CD86, major histocompatibility complex [MHC] II, LC3A/B) involved in antigen processing, presentation, and co-stimulation in antigen-presenting cells (APCs). In contrast, male WT and Cst3-/- mice and cells show no differences in EAE signs or APC function. Further, the sex-dependent effect of CST3 in EAE is sensitive to gonadal hormones. Altogether, we have shown that CST3 has a sex-dependent role in MOG35-55-induced EAE.

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.

Small Heat Shock Proteins, Amyloid Fibrils, and Nicotine Stimulate a Common Immune Suppressive Pathway with Implications for Future Therapies.

The α7 nicotinic acetylcholine receptor (α7nAChR) is central to the anti-inflammatory function of the vagus nerve in a physiological mechanism termed the inflammatory reflex. Studies on the inflammatory reflex have been instrumental for the current development of the field of bioelectronic medicine. An independent investigation of the biological role of αB-crystallin (HspB5), the most abundant gene transcript present in active multiple sclerosis lesions in human brains, also led to α7nAChR. Induction of experimental autoimmune encephalomyelitis (EAE) in HspB5-/- mice results in greater paralytic signs, increased levels of proinflammatory cytokines, and T-lymphocyte activation relative to wild-type animals. Administration of HspB5 was therapeutic in animal models of multiple sclerosis, retinal and cardiac ischemia, and stroke. Structure-activity studies established that residues 73-92 were as potent as the parent protein, but only when it formed amyloid fibrils. Amyloid fibrils and small heat shock proteins (sHsps) selectively bound α7nAChR on peritoneal macrophages (MΦs) and B lymphocytes, converting the MΦs to an immune suppressive phenotype and mobilizing the migration of both cell types from the peritoneum to secondary lymph organs. Here, we review multiple aspects of this work, which may be of interest for developing future therapeutic approaches for multiple sclerosis and other disorders.

Astrocytes and Aging.

By 2050, the aging population is predicted to expand by over 100%. Considering this rapid growth, and the additional strain it will place on healthcare resources because of age-related impairments, it is vital that researchers gain a deeper understanding of the cellular interactions that occur with normal aging. A variety of mammalian cell types have been shown to become compromised with age, each with a unique potential to contribute to disease formation in the aging body. Astrocytes represent the largest group of glial cells and are responsible for a variety of essential functions in the healthy central nervous system (CNS). Like other cell types, aging can cause a loss of normal function in astrocytes which reduces their ability to properly maintain a healthy CNS environment, negatively alters their interactions with neighboring cells, and contribute to the heightened inflammatory state characteristic of aging. The goal of this review article is to consolidate the knowledge and research to date regarding the role of astrocytes in aging. In specific, this review article will focus on the morphology and molecular profile of aged astrocytes, the consequence of astrocyte dysfunction on homeostatic functions during aging, and the role of astrocytes in age-related neurodegenerative diseases.

Biochemically altered myelin triggers autoimmune demyelination.

Although immune attack against central nervous system (CNS) myelin is a central feature of multiple sclerosis (MS), its root cause is unresolved. In this report, we provide direct evidence that subtle biochemical modifications to brain myelin elicit pathological immune responses with radiological and histological properties similar to MS lesions. A subtle myelinopathy induced by abbreviated cuprizone treatment, coupled with subsequent immune stimulation, resulted in lesions of inflammatory demyelination. The degree of myelin injury dictated the resulting immune response; biochemical damage that was too limited or too extensive failed to trigger overt pathology. An inhibitor of peptidyl arginine deiminases (PADs), enzymes that alter myelin structure and correlate with MS lesion severity, mitigated pathology even when administered only during the myelin-altering phase. Moreover, cultured splenocytes were reactive against donor myelin isolates, a response that was substantially muted when splenocytes were exposed to myelin from donors treated with PAD inhibitors. By showing that a primary biochemical myelinopathy can trigger secondary pathological inflammation, "cuprizone autoimmune encephalitis" potentially reconciles conflicting theories about MS pathogenesis and provides a strong rationale for investigating myelin as a primary target for early, preventative therapy.

Murine neutrophils treated with alphaB-crystallin reduce IL-12p40 production by dendritic cells.

Neutrophils are essential in the fight against invading pathogens. They utilize antimicrobial effector mechanisms, such as phagocytosis, release of proteases and other antimicrobial products, robust oxidative bursts and neutrophil extracellular traps to combat infections. Neutrophils also modulate immune responses through the production of eicosanoids, cytokines and chemokines, as well as via direct communication with other immune cells. This system of high-intensity offense against pathogens is exquisitely balanced through regulation to limit damage to host tissue. Unfortunately, the control of neutrophils is not failproof. In cases of sterile injury, autoimmunity and even during an infection, neutrophils can cause tissue destruction and become detrimental to the host. For that reason, there is a need to find means to regulate the aberrant activation of these cells. We found that alphaB-crystallin (αBC), a heat-shock protein known to have anti-inflammatory abilities, affects certain properties of mouse neutrophils that subsequently influence the pro-inflammatory state of antigen-presenting cells (APCs). More specifically, αBC mediated small but significant increases in the levels of IL-10 and matrix metalloproteinase 8, and altered hydrogen peroxide secretion by stimulated neutrophils. Further, the heat-shock protein influenced the communication between neutrophils and dendritic cells by decreasing the production of pro-inflammatory cytokines, specifically IL-12p40, by the APCs. αBC could thus contribute to dampening neutrophil inflammatory responses by impacting the effect of neutrophils on other immune cells.

Chaperone Proteins in the Central Nervous System and Peripheral Nervous System after Nerve Injury.

Injury to axons of the central nervous system (CNS) and the peripheral nervous system (PNS) is accompanied by the upregulation and downregulation of numerous molecules that are involved in mediating nerve repair, or in augmentation of the original damage. Promoting the functions of beneficial factors while reducing the properties of injurious agents determines whether regeneration and functional recovery ensues. A number of chaperone proteins display reduced or increased expression following CNS and PNS damage (crush, transection, contusion) where their roles have generally been found to be protective. For example, chaperones are involved in mediating survival of damaged neurons, promoting axon regeneration and remyelination and, improving behavioral outcomes. We review here the various chaperone proteins that are involved after nervous system axonal damage, the functions that they impact in the CNS and PNS, and the possible mechanisms by which they act.

AlphaB-crystallin regulates remyelination after peripheral nerve injury.

AlphaB-crystallin (αBC) is a small heat shock protein that is constitutively expressed by peripheral nervous system (PNS) axons and Schwann cells. To determine what role this crystallin plays after peripheral nerve damage, we found that loss of αBC impaired remyelination, which correlated with a reduced presence of myelinating Schwann cells and increased numbers of nonmyelinating Schwann cells. The heat shock protein also seems to regulate the cross-talk between Schwann cells and axons, because expected changes in neuregulin levels and ErbB2 receptor expression after PNS injury were disrupted in the absence of αBC. Such dysregulations led to defects in conduction velocity and motor and sensory functions that could be rescued with therapeutic application of the heat shock protein in vivo. Altogether, these findings show that αBC plays an important role in regulating Wallerian degeneration and remyelination after PNS injury.

AlphaB-crystallin expression correlates with aging deficits in the peripheral nervous system.

In an effort to identify factors that contribute to age-related deficits in the undamaged and injured peripheral nervous system (PNS), we noted that Brady and colleagues found that mice null for a small heat shock protein called alphaB-crystallin (αBC) developed abnormalities early in life that are reminiscent of aging pathologies. Because of our observation that αBC protein levels markedly reduce as wild-type mice age, we investigated whether the crystallin plays a role in modulating age-related deficits in the uninjured and damaged PNS. We show here that the presence of αBC correlates with maintenance of myelin sheath thickness, reducing macrophage presence, sustaining lipid metabolism, and promoting remyelination following peripheral nerve injury in an age-dependent manner. More specifically, animals null for αBC displayed a higher frequency of thinly myelinated axons, enhanced presence of Iba1+ macrophages, and fewer immunoreactive profiles of the cholesterol biosynthesis enzyme, squalene monooxygenase, before and after sciatic nerve crush injury. These findings thus suggest that αBC plays a protective and beneficial role in the aging PNS.

Incidence, prevalence, and occurrence rate of infection among adults hospitalized after traumatic brain injury: study protocol for a systematic review and meta-analysis.

BACKGROUND: Infection occurs commonly among patients hospitalized after traumatic brain injury (TBI) and has been associated with increased intensive care unit and hospital lengths of stay and an elevated risk of poor neurological outcome and mortality. However, as many relevant published studies to date have varied in the type and severity of TBI among included patients as well as in their design (randomized versus non-randomized), risk of bias, and setting (hospital ward versus intensive care unit), their reported estimates of infection occurrence vary considerably. Thus, the purpose of this systematic review and meta-analysis is to estimate the incidence, prevalence, and occurrence rate of infection among patients hospitalized after TBI. METHODS/DESIGN: We will search electronic bibliographic databases (MEDLINE, EMBASE, PubMed, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Scopus, Web of Science, the Cochrane Central Register of Controlled Trials (CENTRAL), and the Cochrane Database of Systematic Reviews) from their first available date as well as personal files, reference lists of included articles, and conference proceedings. Two investigators will independently screen titles and abstracts and select cohort studies, cross-sectional studies, and randomized controlled trials involving adults hospitalized after TBI that reported estimates of cumulative incidence, incidence rate, prevalence, or occurrence rate of infection for inclusion in the systematic review. These investigators will also independently extract data and assess risk of bias. We will exclude studies with fewer than ten patients; experimental groups allocated to treatment with antibiotics, glucocorticoids, immunosuppressants, barbiturates, or hypothermia; and studies focused on military/combat-related TBI. Pooled estimates of cumulative incidence, incidence rate, prevalence, and occurrence rate will be calculated using random effects models. We will also calculate I2 and Cochran Q statistics to assess for inter-study heterogeneity and conduct stratified analyses and univariate meta-regression to determine the influence of pre-defined study-level covariates on our pooled estimates. DISCUSSION: This study will compile the world literature regarding the epidemiology of infection among adults hospitalized after TBI. A better understanding of the role of infection will be helpful in the development of guidelines for patient management. This protocol has been registered in the PROSPERO International Prospective Register of Systematic Reviews (ID: CRD42013005146).

Altered cognitive-emotional behavior in early experimental autoimmune encephalitis--cytokine and hormonal correlates.

Multiple sclerosis (MS) is often associated with co-morbid behavioural and cognitive impairments; however the presence of these symptoms does not necessarily correlate with neurological damage. This suggests that an alternate mechanism may subserve these impairments relative to motor deficits. We investigated whether these abnormalities could be studied in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced EAE mice, no motor deficits were observed until d9 after immunization. This enabled us to carry out a series of neurobehavioral tests during the presymptomatic stage, between d6 and d8 post-immunization. EAE mice spent more time in the outer zone in an open field test and in the closed arms of an elevated plus maze and, showed decreased latency for immobility in the tail suspension and forced swim tests and reduced social interaction compared with controls. These results are indicative of anxiety- and depression- like behavior. In addition, EAE mice appeared to exhibit memory impairment compared to controls based on their reduced time spent in the target quadrant in the Morris water maze and their faster memory extinction in the fear conditioning test. No demyelination, microglial activation or astrogliosis was observed in the brain at this early stage. Transcript analysis by RT-PCR from d6 to d8 brain revealed elevated interleukin (IL)-1ß and TNF-α in the hypothalamus but not in the amygdala or hippocampus of EAE mice. Lastly, plasma corticosterone levels increased in EAE mice compared to controls. In conclusion, emotional and cognitive deficits are observed in EAE prior to demyelination and are associated with elevated IL-1ß and TNF-α in the hypothalamus and changes in the hypothalamic-pituitary-adrenal axis.

CRYAB modulates the activation of CD4+ T cells from relapsing-remitting multiple sclerosis patients.

BACKGROUND: Suppression of activation of pathogenic CD4(+) T cells is a potential therapeutic intervention in multiple sclerosis (MS). We previously showed that a small heat shock protein, CRYAB, reduced T cell proliferation, pro-inflammatory cytokine production and clinical signs of experimental allergic encephalomyelitis, a model of MS. OBJECTIVE: We assessed whether the ability of CRYAB to reduce the activation of T cells translated to the human disease. METHODS: CD4(+) T cells from healthy controls and volunteers with MS were activated in vitro in the presence or absence of a CRYAB peptide (residues 73-92). Parameters of activation (proliferation rate, cytokine secretion) and tolerance (anergy, activation-induced cell death, microRNAs) were evaluated. RESULTS: The secretion of pro-inflammatory cytokines by CD4(+) T cells was decreased in the presence of CRYAB in a subset of relapsing-remitting multiple sclerosis (RRMS) participants with mild disease severity while no changes were observed in healthy controls. Further, there was a correlation for higher levels of miR181a microRNA, a marker upregulated in tolerant CD8(+) T cells, in CD4(+) T cells of MS patients that displayed suppressed cytokine production (responders). CONCLUSION: CRYAB may be capable of suppressing the activation of CD4(+) T cells from a subset of RRMS patients who appear to have less disability but similar age and disease duration.

Characterization of migration parameters on peripheral and central nervous system T cells following treatment of experimental allergic encephalomyelitis with CRYAB.

CRYAB, a small heat shock protein, was previously shown to decrease neuroinflammation in experimental allergic encephalomyelitis (EAE). We investigated whether the expression of cell adhesion molecules and chemokine receptors on peripheral and spinal cord T cells, that could possibly affect their migration to the central nervous system, was altered following EAE CRYAB treatment. Less LFA-1+ lymphocytes and lower levels of iTAC, MCP-5 and MIG were observed in spinal cords of CRYAB-injected EAE animals. In addition, fewer blood T cells expressed CCR6, CXCR4 and CCR7 and in vivo-derived CRYAB EAE CD4+ lymphocytes were less migratory towards a MIP-3alpha gradient in vitro.

Piet Mondrian's trees and the evolution in understanding multiple sclerosis, Charcot Prize Lecture 2011.

Four questions were posed about multiple sclerosis (MS) at the 2011 Charcot Lecture, Oct. 22, 2011. 1. The Male/Female Disparity: Why are women developing MS so much more frequently than men? 2. Neuronal and Glial Protection: Are there guardian molecules that protect the nervous system in MS? 3. Predictive Medicine: With all the approved drugs, how can we rationally decide which one to use? 4. The Precise Scalpel vs. the Big Hammer for Therapy: Is antigen-specific therapy for demyelinating disease possible? To emphasize how our views on the pathogenesis and treatment of MS are evolving, and given the location of the talk in Amsterdam, Piet Mondrian's progressive interpretations of trees serve as a heuristic.

Chaperone activity of small heat shock proteins underlies therapeutic efficacy in experimental autoimmune encephalomyelitis.

To determine whether the therapeutic activity of αB crystallin, small heat shock protein B5 (HspB5), was shared with other human sHsps, a set of seven human family members, a mutant of HspB5 G120 known to exhibit reduced chaperone activity, and a mycobacterial sHsp were expressed and purified from bacteria. Each of the recombinant proteins was shown to be a functional chaperone, capable of inhibiting aggregation of denatured insulin with varying efficiency. When injected into mice at the peak of disease, they were all effective in reducing the paralysis in experimental autoimmune encephalomyelitis. Additional structure activity correlations between chaperone activity and therapeutic function were established when linear regions within HspB5 were examined. A single region, corresponding to residues 73-92 of HspB5, forms amyloid fibrils, exhibited chaperone activity, and was an effective therapeutic for encephalomyelitis. The linkage of the three activities was further established by demonstrating individual substitutions of critical hydrophobic amino acids in the peptide resulted in the loss of all of the functions.