Axon Density Research Articles

Relationships between abdominal adipose tissue and neuroinflammation with diffusion basis spectrum imaging in midlife obesity

AbstractObjectiveThis study investigated how obesity, BMI ≥ 30 kg/m2, abdominal adiposity, and systemic inflammation relate to neuroinflammation using diffusion basis spectrum imaging.MethodsWe analyzed data from 98 cognitively normal midlife participants (mean age: 49.4 [SD 6.2] years; 34 males [34.7%]; 56 with obesity [57.1%]). Participants underwent brain and abdominal magnetic resonance imaging (MRI), blood tests, and amyloid positron emission tomography (PET) imaging. Abdominal visceral and subcutaneous adipose tissue (VAT and SAT, respectively) was segmented, and Centiloids were calculated. Diffusion basis spectrum imaging parameter maps were created using an in‐house script, and tract‐based spatial statistics assessed white matter differences in high versus low BMI values, VAT, SAT, insulin resistance, systemic inflammation, and Centiloids, with age and sex as covariates.ResultsObesity, high VAT, and high SAT were linked to lower axial diffusivity, reduced fiber fraction, and increased restricted fraction in white matter. Obesity was additionally associated with higher hindered fraction and lower fractional anisotropy. Also, individuals with high C‐reactive protein showed lower axial diffusivity. Higher restricted fraction correlated with continuous BMI and SAT particularly in male individuals, whereas VAT effects were similar in male and female individuals.ConclusionsThe findings suggest that, at midlife, obesity and abdominal fat are associated with reduced brain axonal density and increased inflammation, with visceral fat playing a significant role in both sexes.image

Non-resolving neuroinflammation regulates axon regeneration in chronic spinal cord injury.

Chronic spinal cord injury (SCI) lesions retain increased densities of microglia and macrophages. In acute SCI, macrophages induce growth cone collapse and facilitate axon retraction away from lesion boundaries. Little is known about the role of sustained inflammation in chronic SCI, or whether chronic inflammation affects regeneration. We used the CSF1R inhibitor, PLX-5622, to deplete microglia and macrophages months after complete crush SCI in female mice. Transcriptional analyses revealed a significant inflammatory depletion within chronic SCI lesions after PLX-5622 treatment. Both transcriptional analyses and immunohistochemistry revealed that Iba1+ cells repopulate to pre-depleted densities after treatment removal. Neuronal-enriched transcripts were significantly elevated in mice after inflammatory repopulation, but no significant effects were observed with inflammatory depletion alone. Axon densities also significantly increased within the lesion after PLX-5622 treatment and after repopulation. To better examine the effect of chronic inflammation on axon regeneration, we tested PLX-5622 treatment in neuronal-specific PTEN knockout (PTEN-KO) mice. PTEN-KO was delivered using spinal injections of retrogradely transported adeno-associated viruses (AAVrg's). PTEN-KO did not further increase axon densities within the lesion beyond effects induced by PLX-5622. Axons that grew within the lesion were histologically identified as 5-HT+ and CGRP+, both of which are not robustly transduced by AAVrg's. Our work identified that increased macrophage/microglia densities in the chronic SCI environment may be actively retained by homeostatic mechanisms likely affiliated with a sustained elevated expression of CSF1 and other chemokines. Finally, we identify a novel role of sustained inflammation as a prospective barrier to axon regeneration in chronic SCI.Significance Statement Inflammatory macrophages infiltrate the spinal cord after a spinal cord injury (SCI) and remain at elevated densities around the lesion chronically. Little is known about the consequences of sustained spinal inflammation in chronic SCI, but it is known that macrophages impair regeneration early after SCI. We hypothesized that persistent neuroinflammation may function as a sustained barrier that chronically impairs axon regeneration. Our work identifies that the chronic SCI environment actively maintains increased densities of macrophages and implicates sustained inflammation as a persistent barrier to repair. Findings from our work change our understanding of the maintenance of non-resolving inflammation in chronic SCI and identifies a need to focus on overcoming chronic inflammatory barriers that limit repair after neurotrauma.

Cytosolic Phospholipase A2 in Infiltrating Monocyte-Derived Macrophages Does Not Impair Recovery After Spinal Cord Injury in Female Mice.

Spinal cord injury (SCI) leads to permanent motor and sensory loss that is exacerbated by intraspinal inflammation that persists months to years after injury. After SCI, monocyte-derived macrophages (MDMs) infiltrate the lesion to aid in myelin-rich debris clearance. During debris clearance, MDMs adopt a proinflammatory phenotype that exacerbates neurodegeneration and hinders recovery. The underlying cause of the lipid-mediated MDM phenotype shift is unclear. Our previous work suggests that cytosolic phospholipase A2 (cPLA2) plays a role in the proinflammatory potentiating effect of myelin on macrophages in vitro . Cytosolic phospholipase A2 (cPLA2) frees arachidonic acid from phospholipids, generating eicosanoids that play an important role in inflammation, immunity, and host defense. cPLA2 is expressed in macrophages along with multiple other cell types after SCI, and cPLA2 inhibition has been reported to both reduce and exacerbate secondary injury pathology recovery. The role of cPLA2 in MDMs after SCI is not fully understood. We hypothesize that cPLA2 activation in MDMs after SCI contributes to secondary injury. Here, we report that cPLA2 plays an important role in the myelin-induced inflammatory macrophage phenotype in vitro using macrophages derived from cPLA2 knockout bone marrow. Furthermore, to investigate the role of cPLA2 in MDMs after SCI, we generated female bone marrow chimeras using cPLA2 knock-out donors and assessed locomotor recovery using the Basso Mouse Scale (BMS), CatWalk gait analysis system, and horizontal ladder task over six weeks. We also evaluated tissue sparing and intralesional axon density six weeks after injury. cPLA2 KO chimeras did not display altered locomotor recovery or tissue pathology after SCI compared to WT chimera controls. These data suggest that although cPLA2 plays a critical role in myelin-mediated potentiation of proinflammatory macrophage activation in vitro , it may not contribute to secondary injury pathology in vivo after SCI.

Cell class-specific long-range axonal projections of neurons in mouse whisker-related somatosensory cortices.

Long-range axonal projections of diverse classes of neocortical excitatory neurons likely contribute to brain-wide interactions processing sensory, cognitive and motor signals. Here, we performed light-sheet imaging of fluorescently labeled axons from genetically defined neurons located in posterior primary somatosensory barrel cortex and supplemental somatosensory cortex. We used convolutional networks to segment axon-containing voxels and quantified their distribution within the Allen Mouse Brain Atlas Common Coordinate Framework. Axonal density was analyzed for different classes of glutamatergic neurons using transgenic mouse lines selectively expressing Cre recombinase in layer 2/3 intratelencephalic projection neurons (Rasgrf2-dCre), layer 4 intratelencephalic projection neurons (Scnn1a-Cre), layer 5 intratelencephalic projection neurons (Tlx3-Cre), layer 5 pyramidal tract projection neurons (Sim1-Cre), layer 5 projection neurons (Rbp4-Cre), and layer 6 corticothalamic neurons (Ntsr1-Cre). We found distinct axonal projections from the different neuronal classes to many downstream brain areas, which were largely similar for primary and supplementary somatosensory cortices. Functional connectivity maps obtained from optogenetic activation of sensory cortex and wide-field imaging revealed topographically organized evoked activity in frontal cortex with neurons located more laterally in somatosensory cortex signaling to more anteriorly located regions in motor cortex, consistent with the anatomical projections. The current methodology therefore appears to quantify brain-wide axonal innervation patterns supporting brain-wide signaling.

Cell class-specific long-range axonal projections of neurons in mouse whisker-related somatosensory cortices

Long-range axonal projections of diverse classes of neocortical excitatory neurons likely contribute to brain-wide interactions processing sensory, cognitive and motor signals. Here, we performed light-sheet imaging of fluorescently labeled axons from genetically defined neurons located in posterior primary somatosensory barrel cortex and supplemental somatosensory cortex. We used convolutional networks to segment axon-containing voxels and quantified their distribution within the Allen Mouse Brain Atlas Common Coordinate Framework. Axonal density was analyzed for different classes of glutamatergic neurons using transgenic mouse lines selectively expressing Cre recombinase in layer 2/3 intratelencephalic projection neurons (Rasgrf2-dCre), layer 4 intratelencephalic projection neurons (Scnn1a-Cre), layer 5 intratelencephalic projection neurons (Tlx3-Cre), layer 5 pyramidal tract projection neurons (Sim1-Cre), layer 5 projection neurons (Rbp4-Cre), and layer 6 corticothalamic neurons (Ntsr1-Cre). We found distinct axonal projections from the different neuronal classes to many downstream brain areas, which were largely similar for primary and supplementary somatosensory cortices. Functional connectivity maps obtained from optogenetic activation of sensory cortex and wide-field imaging revealed topographically organized evoked activity in frontal cortex with neurons located more laterally in somatosensory cortex signaling to more anteriorly located regions in motor cortex, consistent with the anatomical projections. The current methodology therefore appears to quantify brain-wide axonal innervation patterns supporting brain-wide signaling.

Anti-inflammatory and remyelinating effects of fexagratinib in experimental multiple sclerosis.

FGF, VEGFR-2 and CSF1R signalling pathways play a key role in the pathogenesis of multiple sclerosis (MS). Selective inhibition of FGFR by infigratinib in MOG35-55-induced experimental autoimmune encephalomyelitis (EAE) prevented severe first clinical episodes by 40%; inflammation and neurodegeneration were reduced, and remyelination was enhanced. Multi-kinase inhibition of FGFR1-3, CSFR and VEGFR-2 by fexagratinib (formerly known as AZD4547) may be more efficient in reducing inflammation, neurodegeneration and regeneration in the disease model. Female C57BL/6J mice were treated with fexagratinib (6.25 or 12.5 mg·kg-1) orally or placebo over 10 days either from time of EAE induction (prevention experiment) or onset of symptoms (suppression experiment). Effects on inflammation, neurodegeneration and remyelination were assessed at the peak of the disease (Day 18/20 post immunization) and the chronic phase of EAE (Day 41/42). In the prevention experiment, treatment with 6.25 or 12.5 mg·kg-1 fexagratinib prevented severe first clinical episodes by 66.7% or 84.6% respectively. Mice treated with 12.5 mg·kg-1 fexagratinib hardly showed any symptoms in the chronic phase of EAE. In the suppression experiment, fexagratinib resulted in a long-lasting reduction of severe symptoms by 91 or 100%. Inflammation and demyelination were reduced, and axonal density, numbers of oligodendrocytes and their precursor cells, and remyelinated axons were increased by both experimental approaches. Multi-kinase inhibition by fexagratinib in a well-tolerated dose of 1 mg·kg-1 in humans may be a promising approach to reduce inflammation and neurodegeneration, to slow down disease progression and support remyelination in patients.

Ultra-high-resolution mapping of myelin and g-ratio in a panel of Mbp enhancer-edited mouse strains using microstructural MRI

Non-invasive myelin water fraction (MWF) and g-ratio mapping using microstructural MRI have the potential to offer critical insights into brain microstructure and our understanding of neuroplasticity and neuroinflammation. By leveraging a unique panel of variably hypomyelinating mouse strains, we validated a high-resolution, model-free image reconstruction method for whole-brain MWF mapping. Further, by employing a bipolar gradient echo MRI sequence, we achieved high spatial resolution and robust mapping of MWF and g-ratio across the whole mouse brain. Our regional white matter-tract specific analyses demonstrated a graded decrease in MWF in white matter tracts which correlated strongly with myelin basic protein gene (Mbp) mRNA levels. Using these measures, we derived the first sensitive calibrations between MWF and Mbp mRNA in the mouse. Minimal changes in axonal density supported our hypothesis that observed MWF alterations stem from hypomyelination. Overall, our work strongly emphasizes the potential of non-invasive, MRI-derived MWF and g-ratio modeling for both preclinical model validation and ultimately translation to humans.

AxonFinder: Automated segmentation of tumor innervating neuronal fibers.

Neurosignaling is increasingly recognized as a critical factor in cancer progression, where neuronal innervation of primary tumors contributes to the disease's advancement. This study focuses on segmenting individual axons within the prostate tumor microenvironment, which have been challenging to detect and analyze due to their irregular morphologies. We present a novel deep learning-based approach for the automated segmentation of axons, AxonFinder, leveraging a U-Net model with a ResNet-101 encoder, based on a multiplexed imaging approach. Utilizing a dataset of whole-slide images from low-, intermediate-, and high-risk prostate cancer patients, we manually annotated axons to train our model, achieving significant accuracy in detecting axonal structures that were previously hard to segment. Our analysis includes a comprehensive assessment of axon density and morphological features across different CAPRA-S prostate cancer risk categories, providing insights into the correlation between tumor innervation and cancer progression. Our paper suggests the potential utility of neuronal markers in the prognostic assessment of prostate cancer in aiding the pathologist's assessment of tumor sections and advancing our understanding of neurosignaling in the tumor microenvironment.

Effect of vascularized jejunal conduit flap on peripheral nerve regeneration in rats.

In the literature, almost all of the nerve conduits proposed for obtaining better nerve recovery were applied as graft materials. In this study, we aimed to propose a new nerve conduit model with a flap pattern and evaluate the effect of a pedicled vascularized jejunal flap on nerve regeneration after wrapping it around a sciatic nerve. A total of 90 Wistar albino rats were randomly divided into nine groups with 10 rats in each. The first three groups constituted the control groups, whereas Groups 4-6 were the jejunum conduit (JC)-applied groups. A mucosa-resected JC (MRJC) was applied in Groups 7 and 8. Epineurial neurorrhaphy was performed in Groups 1, 4, and 7; repair with a nerve graft was applied in Groups 2, 5, and 8; and a 1-cm-long nerve defect was created in Groups 3, 6, and 9. After 2 months of follow-up, nerve regeneration was assessed by statistical analyses of the Sciatic Functional Index (SFI) and histopathological evaluation. The MRJC groups had significantly better results in terms of SFI (p = 0.005). Statistical differences in axonal degeneration, axonal density, myelination, and disorganization were found between all control groups and MRJC groups (p = 0.022, p = 0.001, p = 0.001, and p = 0.039, respectively). In this study, the feasibility of wrapping around the nerve repair zones of pedicled autologous flaps designed in a tubular fashion was observed in a small rat model. The findings must be further validated with larger animals before clinical testing.

Effect of 28 days treatment of baricitinib on mechanical allodynia, osteopenia, and loss of nerve fibers in an experimental model of type-1 diabetes mellitus.

Type-1 diabetes mellitus (T1DM) is associated with numerous health problems, including peripheral neuropathy, osteoporosis, and bone denervation, all of which diminish quality of life. However, there are relatively few therapies to treat these T1DM-related complications. Recent studies have shown that Janus kinase (JAK) inhibitors reverse aging- and rheumatoid arthritis-induced bone loss and reduce pain associated with peripheral nerve injuries, and rheumatoid arthritis. Thus, we assessed whether a JAK1/JAK2 inhibitor, baricitinib, ameliorates mechanical pain sensitivity (a measure of peripheral neuropathy), osteoporosis, and bone denervation in the femur of mice with T1DM. Female ICR mice (13 weeks old) received five daily administrations of streptozotocin (ip, 50mg/kg) to induce T1DM. At thirty-one weeks of age, mice were treated with baricitinib (po; 40mg/kg/bid; for 28 days) or vehicle. Mechanical sensitivity was evaluated at 30, 33, and 35 weeks of age on the plantar surface of the right hind paw. At the end of the treatment, mice were sacrificed, and lower extremities were harvested for microcomputed tomography and immunohistochemistry analyses. Mice with T1DM exhibited greater blood glucose levels, hind paw mechanical hypersensitivity, trabecular bone loss, and decreased density of calcitonin gene-related peptide-positive and tyrosine hydroxylase-positive axons within the marrow of the femoral neck compared to control mice. Baricitinib treatment significantly reduced mechanical hypersensitivity and ameliorated sensory and sympathetic denervation at the femoral neck, but it did not reverse trabecular bone loss. Our findings suggest that baricitinib may represent a new therapeutic alternative to treat T1DM-induced peripheral neuropathy and bone denervation.

Value of Spinal Cord Diffusion Imaging and Tractography in Providing Predictive Factors for Tumor Resection in Patients with Intramedullary Tumors: A Pilot Study.

This pilot study aimed to investigate the interest of high angular resolution diffusion imaging (HARDI) and tractography of the spinal cord (SC) in the management of patients with intramedullary tumors by providing predictive elements for tumor resection. Eight patients were included in a prospective study. HARDI images of the SC were acquired using a 3T MRI scanner with a reduced field of view. Opposed phase-encoding directions allowed distortion corrections. SC fiber tracking was performed using a deterministic approach, with extraction of tensor metrics. Then, regions of interest were drawn to track the spinal pathways of interest. HARDI and tractography added value by providing characteristics about the microstructural organization of the spinal white fibers. In patients with SC tumors, tensor metrics demonstrated significant changes in microstructural architecture, axonal density, and myelinated fibers (all, p < 0.0001) of the spinal white matter. Tractography aided in the differentiation of tumor histological types (SC-invaded vs. pushed back by the tumor), and differentiation of the spinal tracts enabled the determination of precise anatomical relationships between the tumor and the SC, defining the tumor resectability. This study underlines the value of using HARDI and tractography in patients with intramedullary tumors, to show alterations in SC microarchitecture and to differentiate spinal tracts to establish predictive factors for tumor resectability.

A potential treatment for erectile dysfunction: Effect of platelet-rich plasma administration on axon and collagen regeneration in cavernous nerve injury.

Recent studies highlighted the role of platelet-rich plasma (PRP) in progenitor cell homing, migration, and nerve cell regeneration while also inhibiting fibrosis and apoptosis in cavernous nerve injury (CNI). The aim of this study was to investigate the effect of PRP administration on axon and collagen regeneration in CNI. A true experimental study using a post-test-only control group design was conducted. Twenty-five male Wistar rats (Rattus norvegicus), weighing 200-300 grams, were divided into five groups: two control groups (sham procedure and negative control), and three experimental groups receiving local PRP, intraperitoneal PRP, and a combination of local and intraperitoneal PRP. The cavernous nerve was injured with a hemostasis clamp for one minute before 200 µL of 200 PRP was injected locally, intraperitoneally, or both, depending on the group. After four weeks, the rats were euthanized, tissue segments (2 mm) from each cavernous nerve and mid-penis were collected and analyzed for collagen density, axon diameter, and number of myelinated axons. Our study found that collagen growth was slower in CNI group without PRP (sham procedure) compared to all PRP groups (local, intraperitoneal, and combination). The intraperitoneal PRP group had the highest collagen density at 5.62 µm; however, no significant difference was observed in collagen density among all groups (p=0.056). Similar axon diameter was found across the groups, with no statistically significant difference observed (p=0.856). In the number of myelinated axons, a significant difference was found among all groups with significantly more axons in local PRP and combined local and intraperitoneal PRP groups compared to others (p=0.026). In conclusion, PRP administration improved the number of myelinated axons in CNI, suggesting PRP role in CNI regeneration and the potential for an innovative approach to treating erectile dysfunction associated with CNI.

Tempol, a Superoxide Dismutase Mimetic, Inhibits Wallerian Degeneration Following Spinal Cord Injury by Preventing Glutathione Depletion and Aldose Reductase Activation.

Spinal cord contusion injury results in Wallerian degeneration of spinal cord axonal tracts, which are necessary for locomotor function. Axonal swelling and loss of axonal density at the contusion site, characteristic of Wallerian degeneration, commence within hours of injury. Tempol, a superoxide dismutase mimetic, was previously shown to reduce the loss of spinal cord white matter and improve locomotor function in an experimental model of spinal cord contusion, suggesting that tempol treatment might inhibit Wallerian degeneration of spinal cord axons. Here, we report that tempol partially inhibits Wallerian degeneration, resulting in improved locomotor recovery. We previously reported that Wallerian degeneration is reduced by inhibitors of aldose reductase (AR), which converts glucose to sorbitol in the polyol pathway. We observed that tempol inhibited sorbitol production in the injured spinal cord to the same extent as the AR inhibitor, sorbinil. Tempol also prevented post-contusion upregulation of AR (AKR1B10) protein expression within degenerating axons, as previously observed for AR inhibitors. Additionally, we hypothesized that tempol inhibits axonal degeneration by preventing loss of the glutathione pool due to polyol pathway activity. Consistent with our hypothesis, tempol treatment resulted in greater glutathione content in the injured spinal cord, which was correlated with increased expression and activity of gamma glutamyl cysteine ligase (γGCL; EC 6.3.2.2), the rate-limiting enzyme for glutathione synthesis. Administration of the γGCL inhibitor buthionine sulfoximine abolished all observed effects of tempol administration. Together, these results support a pathological role for polyol pathway activation in glutathione depletion, resulting in Wallerian degeneration after spinal cord injury (SCI). Interestingly, methylprednisolone, oxandrolone, and clenbuterol, which are known to spare axonal tracts after SCI, were equally effective in inhibiting polyol pathway activation. These results suggest that prevention of AR activation is a common target of many disparate post-SCI interventions.

Treatment of vascular dementia in female rats with AV-001, an Angiopoietin-1 mimetic peptide, improves cognitive function.

Vascular dementia (VaD) is a complex neurodegenerative disorder. We previously found that treatment of VaD in middle-aged male rats subjected to multiple microinfarction (MMI) with AV-001, a Tie2 receptor agonist, significantly improves cognitive function. Age and sex affect the development and response of VaD to therapeutic intervention. Thus, the present study investigated the therapeutic effect of AV-001 on VaD in aged female rats subjected to MMI. Female 18-month-old Wistar rats were subjected to MMI by injecting either 1,000 (low dose, LD-MMI) or 6,000 (high dose, HD-MMI) cholesterol crystals of size 70-100 μm into the right internal carotid artery. AV-001 (1 μg/Kg, i.p.) was administered once daily after MMI for 1 month, with treatment initiated 1 day after MMI. A battery of behavioral tests to examine sensorimotor and cognitive functions was performed at 21-28 days after MMI. All rats were sacrificed at 1 month after MMI. Aged female rats subjected to LD-MMI exhibit severe neurological deficits, memory impairment, and significant white matter (WM) and oligodendrogenesis injury in the corpus callosum compared with control rats. HD-MMI in aged female rats induces significant anxiety- and depression-like behaviors, which were not detected in LD-MMI aged female rats. Also, HD-MMI induces significantly increased WM injury compared to LD-MMI. AV-001 treatment of LD-MMI and HD-MMI increases oligodendrogenesis, myelin and axon density in the corpus callosum and striatal WM bundles, promotes WM integrity and attenuates neurological and cognitive deficits. Additionally, both LD-MMI and HD-MMI rats exhibit a significant increase, while AV-001 significantly decreases the levels of inflammatory factors in the cerebrospinal fluid (CSF). MMI reduces oligodendrogenesis, and induces demyelination, axonal injury and WM injury, and causes memory impairment, while HD-MMI induces increased WM injury and further depression-like behaviors compared to LD-MMI rats. AV-001 has a therapeutic effect on aged female rats with MMI by reducing WM damage and improving neuro-cognitive outcomes.

Diffusion Basis Spectrum Imaging of White Matter in Schizophrenia and Bipolar Disorder.

Multiple studies point to the role of neuroinflammation in the pathophysiology of schizophrenia (SCZ), however, there have been few in vivo tools for imaging brain inflammation. Diffusion basis spectrum imaging (DBSI) is an advanced diffusion-based MRI method developed to quantitatively assess microstructural alternations relating to neuroinflammation, axonal fiber, and other white matter (WM) pathologies. We acquired one-hour-long high-directional diffusion MRI data from young control (CON, n = 27), schizophrenia (SCZ, n = 21), and bipolar disorder (BPD, n = 21) participants aged 18-30. We applied Tract-based Spatial Statistics (TBSS) to allow whole-brain WM analyses and compare DBSI-derived isotropic and anisotropic diffusion measures between groups. Clinical relationships of DBSI metrics with clinical symptoms were assessed across SCZ and control participants. In SCZ participants, we found a generalized increase in DBSI-derived cellularity (a putative marker of neuroinflammation), a decrease in restricted fiber fraction (a putative marker of apparent axonal density), and an increase in extra-axonal water (a putative marker of vasogenic edema) across several WM tracts. There were only minimal WM abnormalities noted in BPD, mainly in regions of the corpus callosum (increase in DTI-derived RD and extra-axonal water). DBSI metrics showed significant partial correlations with psychosis and mood symptoms across groups. Our findings suggest that SCZ involves generalized white matter neuroinflammation, decreased fiber density, and demyelination, which is not seen in bipolar disorder. Larger studies are needed to identify medication-related effects. DBSI metrics could help identify high-risk groups requiring early interventions to prevent the onset of psychosis and improve outcomes.

Augmentation of functional recovery via ROCK/PI3K/AKT pathway by Fasudil Hydrochloride in a rat sciatic nerve transection model

BackgroundsThe functional recovery after peripheral nerve injury remains unsatisfactory. This study aims to perform a comprehensive evaluation of the efficacy of Fasudil Hydrochloride at treating the sciatic nerve transection injury in rats and the mechanism involved. Materials and methodsIn animal experiments, 75 Sprague Dawley rats that underwent transection and repair of the right sciatic nerve were divided into a control, Fasudil, and Fas + LY group, receiving daily intraperitoneal injection of saline, Fasudil Hydrochloride (10 mg/kg), and Fasudil Hydrochloride plus LY294002 (5 mg/kg), respectively. At day 3 after surgery, the expression of ROCK2, p-PI3K, and p-AKT in L4-5 DRG and the lumbosacral enlargement was determined using Western blotting. At day 7 and 14, axon density in the distal stump was evaluated with immunostaining using the anti-Neurofilament-200 antibody. At day 30, retrograde tracing by injecting Fluoro-gold in the distal stump was performed. Three months after surgery, remyelination was analyzed with immostaining using the anti-MPZ antibody and the transmission electron microscope; Moreover, Motion-Evoked Potential, and recovery of sensorimotor functions was evaluated with a neuromonitor, Footprint, Hot Plate and Von Frey Filaments, respectively. Moveover, the Gastrocnemius muscles were weighed, and then underwent H＆E staining, and staining of the neuromuscular junction using α-Bungarotoxin to evaluate the extent of atrophy and degeneration of the endplates in the Gastrocnemius. In vitro, spinal motor neurons (SMNs) and dorsal root ganglia (DRG) were cultured to examine the impact of Fasudil Hydrochloride and LY294002 on the axon outgrowth. ResultsThree days after injury, the expression of ROCK2 increased significantly (P＜0.01), and Fasudil application significantly increased the expression of p-PI3K and p-AKT in L4-6 DRG and the lumbosacral enlargement (P < 0.05). At day 7 and 14 after surgery, a higher axon density could be observed in the Fasudil group(P < 0.05). At day 30 after surgery, a larger number of motor and sensory neurons absorbing Fluoro-gold could be observed in the Fasudil group (P < 0.01) Three months after surgery, a greater thickness of myelin sheath could be observed in the Fasudil group (P < 0.05). The electrophysiological test showed that a larger amplitude of motion-evoked potential could be triggered in the Fasudil group (P < 0.01). Behavioral tests showed that a higher sciatic function index and a lower threshold for reacting to heat and mechanical stimuli could be measured in the Fasudil group. (P < 0.01). The wet weight ratio of the Gastrocnemius muscles and the area of the cross section of its myofibrils were greater in the Fasudil group (P < 0.01), which also demonstrated a higer ratio of axon-endplate connection and a larger size of endplates (P < 0.05). And there were no significant differences for the abovementioned parameters between the control and Fas + LY groups (P＞0.05). In vitro studies showed that Fasudil could significantly promote axon growth in DRG and SMNs, and increase the expression of p-PI3K and p-AKT, which could be abolished by LY294002 (P < 0.05). ConclusionsFasudil can augment axon regeneration and remyelination, and functional recovery after sciatic nerve injury by activating the PI3K/AKT pathway. The translational potential of this articleThe translation potential of this article is that we report for the first time that Fasudil Hydrochloride has a remarkable efficacy at improving axon regeneration and remyelination following a transection injury of the right sciatic nerve in rats through the ROCK/PI3K/AKT pathway, which has a translational potential to be used clinically to treat peripheral nerve injury.

Incorporation of decellularized-ECM in graphene-based scaffolds enhances axonal outgrowth and branching in neuro-muscular co-cultures.

Peripheral nerve and large-scale muscle injuries result in significant disability, necessitating the development of biomaterials that can restore functional deficits by promoting tissue regrowth in an electroactive environment. Among these materials, graphene is favored for its high conductivity, but its low bioactivity requires enhancement through biomimetic components. In this study, we extrusion printed graphene-poly(lactide-co-glycolide) (graphene) lattice scaffolds, aiming to increase bioactivity by incorporating decellularized extracellular matrix (dECM) derived from mouse pup skeletal muscle. We first evaluated these scaffolds using human-induced pluripotent stem cell (hiPSC)-derived motor neurons co-cultured with supportive glia, observing significant improvements in axon outgrowth. Next, we tested the scaffolds with C2C12 mouse and human primary myoblasts, finding no significant differences in myotube formation between dECM-graphene and graphene scaffolds. Finally, using a more complex hiPSC-derived 3D motor neuron spheroid model co-cultured with human myoblasts, we demonstrated that dECM-graphene scaffolds significantly improved axonal expansion towards peripheral myoblasts and increased axonal network density compared to graphene-only scaffolds. Features of early neuromuscular junction formation were identified near neuromuscular interfaces in both scaffold types. These findings suggest that dECM-graphene scaffolds are promising candidates for enhancing neuromuscular regeneration, offering robust support for the growth and development of diverse neuromuscular tissues.

Reduced Gene Expression of KCC2 Accelerates Axonal Regeneration and Reduces Motor Dysfunctions after Tibial Nerve Severance and Suturing

Gamma-aminobutyric acid and glycine (GABA/Gly) are predominantly inhibitory neurotransmitters in the mature central nervous system; however, they mediate membrane potential depolarization during development. These differences in actions depend on intracellular Cl- concentrations ([Cl-]i), which are primarily regulated by potassium chloride cotransporter 2 (KCC2). After nerve injury, KCC2 expression markedly decreases and GABA/Gly mediate depolarization. Following nerve regeneration, KCC2 expression recovers and GABA/Gly become inhibitory, suggesting that KCC2 reduction and GABA/Gly excitation may be crucial for axonal regeneration. To directly clarify their involvement in regeneration, we analyzed recovery processes after tibial nerve severance and suturing between heterozygous KCC2 knockout mice (HT), whose KCC2 levels are halved, and their wild-type littermates (WT). Compared with WT mice, the sciatic functional index—indicating lower limb motor function—was significantly higher until 28 days after operation (D28) in HT mice. Furthermore, at D7, many neurofilament-positive fibers were elongated into the distal part of the sutured nerve in HT mice only, and myelinated axonal density was significantly higher at D21 and D28 in HT animals. Electron microscopy and galanin immunohistochemistry indicated a shorter nerve degeneration period in HT mice. Moreover, a less severe decrease in choline acetyltransferase was observed in HT mice. These results suggest that nerve degeneration and regeneration proceed more rapidly in HT mice, resulting in milder motor dysfunction. Via similar microglial activation, nerve surgery may reduce KCC2 levels more rapidly in HT mice, followed by earlier increased [Cl-]i and longer-lasting GABA/Gly excitation. Taken together, reduced KCC2 may accelerate nerve regeneration via GABA/Gly excitation.

Investigation of the effectiveness of atmospheric pressure cold plasma on sciatic nerve injury in rats.

The aim of this study was to evaluate the efficacy of atmospheric pressure cold plasma jet and plasma activated medium (PAM) on sciatic nerve injury (SNI). Rats were divided into 6 groups (n = 10); group 1 (Sham), group 2 (SNI), group 3 (SNI + Atmospheric pressure cold plasma jet 5 min), group 4 (SNI + Atmospheric pressure cold plasma jet 10 min), group 5 (SNI + PAM 5 min), group 6 (SNI + PAM 10 min). On the 1st, 8th, 15th, 22nd days of the study, atmospheric pressure cold plasma jet was applied to rats in groups 3 and 4, and PAM was applied to rats in groups 5 and 6. Hot plate test was applied to all rats on the same days. On day 28, the experiment was terminated and sciatic nerve tissues were removed for histopathologic evaluations. According to the 4-week average of the hot plate tests, a significant relationship was found between group 2 and group 4 and group 6 (p < 0.05). When evaluated within each week, significant differences were found between group 2 and group 4 in week 1, between group 2 and group 5 and group 6 in week 2, between group 2 and group 4 in week 3, and between group 2 and group 4 and group 6 in week 4 (p < 0.05). As a result of histopathologic analysis, except for the control group, the other groups had similar characteristics in terms of axonal degeneration, periaxonal swelling and axon density. As a result of our study, we found that plasma application showed an improvement in the duration of the hot plate test, but did not show any improvement histopathologically.

Quantitative Analysis of CGRP-IR Afferent Axons in the Mouse Stomach Using Zeiss Arivis Vision4D for Automated Tracing

Gastric nociceptive afferents detect and respond to noxious stimuli by transmitting signals from the stomach to the brain. Calcitonin gene-related peptide (CGRP) is a marker for nociceptive afferents and is crucial to gastric functions, including gastric acid secretion, gut motility, and inflammation. However, the topographical quantitative analysis of CGRP-immunoreactive (CGRP-IR) axons in the whole stomach is challenging due to the large amount of axon innervation in various targets within the tissue. Manual tracing of axons is time-consuming, labor-intensive, and extremely tedious, while currently existing automatic neuron tracing programs often need help with large file sizes or tracing inaccuracies. In this study, we used Zeiss Arivis Vision4D, a novel and sophisticated software with a user-guided machine learning capability, for automated tracing of CGRP-IR axons in flat-mounts of the whole mouse stomach muscular layers and thick serial sections of the antrum-pylorus region (C57BL/6J, male, 2-3 months old, n=7). We utilized a combination of techniques, including the Zeiss M2 Imager for automated scanning, Zen 3.3 (blue edition) for image processing, and Vision4D for stitching the entire stomach flat-mounts and sections, 3D fiber tracing, and density analysis. Our results demonstrated that: 1) Vision4D detected and traced almost all CGRP-IR axons of different gastric targets (blood vessels, muscles, myenteric neurons) in different regions (fundus, corpus, antrum) of the stomach. 2) The x-y-z tracing allowed the visualization of axons in space, portraying its continuous trajectory across layers in the muscular flat mounts (longitudinal muscle, myenteric plexus, circular muscle) and cross sections (muscles, submucosa, mucosa). 3) Vision4D also enabled quantitative morphological and laminar analysis based on target and layer separations. The axon density heatmap generated from the tracing data showed high CGRP-IR axon innervation in the blood vessels. After the removal of blood vessels, dense innervation was seen in the upper fundus and antrum-pylorus followed by corpus, indicating the abundance of CGRP in these regions. 4) Vision4D is a powerful tool in handling large data sizes (hundreds of gigabytes of optical sections in more than 300 image frames per stomach) and outstanding in its capability to track nerve fibers in a highly accurate and time effcient manner. This has paved the way for data batch processing to be accessible, as well as shown that multi-marker tracing of the same tissue sample is attainable. 5) The tracing data will be integrated into a stomach scaffold for a 3D map of CGRP-IR afferent axons. This study represents, for the first time, the automatically traced CGRP-IR axons using whole stomach preparations and highlights the potential of Vision4D in creating axon tracing models in other organs and species. This study is supported by NIH 1 U01 NS113867-01, NIH R15 1R15HL137143-01A1 and Carl ZEISS Company. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.