Infinite Horizon Impactor Research Articles

Neuroprotective role of calreticulin after spinal cord injury in mice

Accumulating evidence suggests that endoplasmic reticulum (ER) stress and unfolded protein response (UPR) are involved in the pathology of spinal cord injury (SCI). To determine the role of the UPR-target molecule in the pathophysiology of SCI, we analyzed the expression and the possible function of calreticulin (CRT), a molecular chaperone in the ER with high Ca2+ binding capacity, in a mouse SCI model. Spinal cord contusion was induced in T9 by using the Infinite Horizon impactor. Quantitative real-time polymerase chain reaction confirmed increase of Calr mRNA after SCI. Immunohistochemistry revealed that CRT expression was observed mainly in neurons in the control (sham operated) condition, while it was strongly observed in microglia/macrophages after SCI. Comparative analysis between wild-type (WT) and Calr+/- mice revealed that the recovery of hindlimb locomotion was reduced in Calr+/- mice, based on the evaluation using the Basso Mouse Scale and inclined-plane test. Immunohistochemistry also revealed more accumulation of immune cells in Calr+/- mice than in WT mice, at the epicenter 3 days and at the caudal region 7 days after SCI. Consistently, the number of damaged neuron was higher in Calr+/- mice at the caudal region 7 days after SCI. These results suggest a regulatory role of CRT in the neuroinflammation and neurodegeneration after SCI.

Degeneration specific NF-L immunoreactivity in spinal cord and brainstem 10 days post-cervical spinal cord contusion

We recently described a panel of novel antibodies to epitopes on the neurofilament light chain (NF-L) which are hidden in healthy neurons and their processes but specifically exposed during neurodegeneration ( doi.org/10.1101/2022.08.27.504533 ). We saw widespread immunopositivity for such antibodies in degenerating and degenerated processes in the rat spinal cord 1-5 days following cervical spinal cord injury (cSCI). Here we aimed to determine if such immunopositivity would persist at 10 days following cSCI. Male Sprague Dawley rats received laminectomy (n=3) or right unilateral contusion at C4 (n=3; 150kdyn; Infinite Horizon Impactor). Tissue was harvested 10 days post-SCI and stained with either Cresyl Violet for routine histology or for immunohistochemistry using mouse monoclonal antibody MCA-6H63, one of several novel antibodies binding to degeneration specific NF-L epitopes (EnCor Biotechnology Inc.). Spinal cord MCA-6H63 immunopositivity was not detected in spinal cords from the laminectomy group. Contusion produced the expected histopathology with ipsilateral grey and white matter disruption and some damage to the contralateral dorsal columns. Punctate labeling for MCA-6H63 was prominent at the lesion epicenter, as well as in the ipsilateral cord rostral and caudal to the injury, consistent with axonal degeneration. Sparse, punctate MCA-6H63 positivity was observed in the contralateral cervical white matter. Qualitatively, MCA-6H63 staining was most robust in the ventral and lateral white matter ipsilateral and caudal to the lesion. This location was consistent with ongoing Wallerian degeneration in prominent spinal tracts including spinothalamic, spinocerebellar, reticulospinal, vestibulospinal and raphespinal. Bilateral MCA-6H63 staining in the dorsal columns was present mostly rostral to the injury. Some MCA-6H63 staining was noted in the medulla, most prominently ipsilateral to the lesion. We conclude that at 10 days post-cSCI this novel NF-L antibody specifically identifies degenerating and degenerated axonal profiles. This is a promising technique in assessing neurodegeneration following SCI and other neurodegenerative diseases. R01HL139708-01A1 (DDF), R01 HL153140-01 (DDF), and EnCor Biotechnology Inc. This is the full abstract presented at the American Physiology Summit 2023 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.

The polarization of microglia and infiltrated macrophages in the injured mice spinal cords: a dynamic analysis.

Following spinal cord injury (SCI), a large number of peripheral monocytes infiltrate into the lesion area and differentiate into macrophages (Mø). These monocyte-derived Mø are very difficult to distinguish from the local activated microglia (MG). Therefore, the term Mø/MG are often used to define the infiltrated Mø and/or activated MG. It has been recognized that pro-inflammatory M1-type Mø/MG play "bad" roles in the SCI pathology. Our recent research showed that local M1 cells are mainly CD45-/lowCD68+CD11b+ in the subacute stage of SCI. Thus, we speculated that the M1 cells in injured spinal cords mainly derived from MG rather than infiltrating Mø. So far, their dynamics following SCI are not yet entirely clear. Female C57BL/6 mice were used to establish SCI model, using an Infinite Horizon impactor with a 1.3 mm diameter rod and a 50 Kdynes force. Sham-operated (sham) mice only underwent laminectomy without contusion. Flow cytometry and immunohistofluorescence were combined to analyze the dynamic changes of polarized Mø and MG in the acute (1 day), subacute (3, 7 and 14 days) and chronic (21 and 28 days) phases of SCI. The total Mø/MG gradually increased and peaked at 7 days post-injury (dpi), and maintained at high levels 14, 21 and 28 dpi. Most of the Mø/MG were activated, and the Mø increased significantly at 1 and 3 dpi. However, with the pathological process, activated MG increased nearly to 90% at 7, 14, 21 and 28 dpi. Both M1 and M2 Mø were increased significantly at 1 and 3 dpi. However, they decreased to very low levels from 7 to 28 dpi. On the contrary, the M2-type MG decreased significantly following SCI and maintained at a low level during the pathological process.

Optimal Ratio of Wnt3a Expression in Human Mesenchymal Stem Cells Promotes Axonal Regeneration in Spinal Cord Injured Rat Model.

ObjectiveThrough our previous clinical trials, the demonstrated therapeutic effects of MSC in chronic spinal cord injury (SCI) were found to be not sufficient. Therefore, the need to develop stem cell agent with enhanced efficacy is increased. We transplanted enhanced Wnt3a-secreting human mesenchymal stem cells (hMSC) into injured spines at 6 weeks after SCI to improve axonal regeneration in a rat model of chronic SCI. We hypothesized that enhanced Wnt3a protein expression could augment neuro-regeneration after SCI. MethodsThirty-six Sprague-Dawley rats were injured using an Infinite Horizon (IH) impactor at the T9–10 vertebrae and separated into five groups : 1) phosphate-buffered saline injection (injury only group, n=7); 2) hMSC transplantation (MSC, n=7); 3) hMSC transfected with pLenti vector (without Wnt3a gene) transplantation (pLenti-MSC, n=7); 4) hMSC transfected with Wnt3a gene transplantation (Wnt3a-MSC, n=7); and 5) hMSC transfected with enhanced Wnt3a gene (1.7 fold Wnt3a mRNA expression) transplantation (1.7 Wnt3a-MSC, n=8). Six weeks after SCI, each 5×105 cells/15 µL at 2 points were injected using stereotactic and microsyringe pump. To evaluate functional recovery from SCI, rats underwent Basso-Beattie-Bresnahan (BBB) locomotor test on the first, second, and third days post-injury and then weekly for 14 weeks. Axonal regeneration was assessed using growth-associated protein 43 (GAP43), microtubule-associated protein 2 (MAP2), and neurofilament (NF) immunostaining. ResultsFourteen weeks after injury (8 weeks after transplantation), BBB score of the 1.7 Wnt3a-MSC group (15.0±0.28) was significantly higher than that of the injury only (10.0±0.48), MSC (12.57±0.48), pLenti-MSC (12.42±0.48), and Wnt3a-MSC (13.71±0.61) groups (p<0.05). Immunostaining revealed increased expression of axonal regeneration markers GAP43, MAP2, and NF in the Wnt3a-MSC and 1.7 Wnt3a-MSC groups. ConclusionOur results showed that enhanced gene expression of Wnt3a in hMSC can potentiate axonal regeneration and improve functional recovery in a rat model of chronic SCI.

Sigma 1 receptor agonist cutamesine promotes plasticity of serotonergic boutons in lumbar enlargement in spinal cord injured rats

Cutamesine, a sigma-1 receptor agonist, functions in both neuroprotection and neurite outgrowth. We assessed the therapeutic effects of cutamesine in a rodent spinal cord injury (SCI) model to demonstrate pre-clinical proof-of-concept. First of all, in order to determine optimal cutamesine dose, cutamesine was administered to normal rats and BDNF protein levels in the lumbar spinal cord were assessed by Western blot. Next, for the SCI model, spinal cords of adult female Sprague-Dawley rats were contused using an Infinite Horizon Impactor. Two weeks post-injury, rats were randomly assigned to receive daily subcutaneous injections of either cutamesine (3.0 mg/kg/day) or saline (as a control) for another two weeks. Immunohistochemistry for BDNF and 5-HT was assessed at four and twelve weeks post-injury in the lumbar spinal cord. Locomotor function was assessed weekly using the BBB locomotor scale until twelve weeks after SCI and CatWalk XT 10.5 gait analysis was conducted at twelve weeks after SCI. In normal rats, cutamesine treatment (3.0 mg/kg/day) significantly up-regulated BDNF expression in the lumbar spinal cord. In SCI rats, cutamesine treatment (3.0 mg/kg/day) significantly increased the fluorescence intensity of neuronal BDNF and serotonin boutons in the injured spinal cord compared to saline. However, cutamesine treatment did not promote significant locomotor recovery. Recent work indicates that cutamesine treatment alone did not promote locomotor recovery in spite of immunohistological changes. Future work will explore the influence of combining cutamesine with other treatment promoting plasticity (e.g. rehabilitative training) in SCI rats.

Development of a Rodent Spinal Cord Injury Model Permissive to Study the Cardiovascular Effects of Rehabilitation Approaches Designed to Induce Neuroplasticity

Background and Aims Spinal cord injury (SCI) induced dysregulation of the cardiovascular (CV) system occurs primarily due to altered descending control over the sympathetic preganglionic neurons. Within the somatic nervous system, the development of pre-clinical models of SCI has provided researchers with a powerful tool to study the effects of neuroplasticity on functional outcomes. To date, a similar pre-clinical model that spares sufficient descending sympathetic pathways to enable the study of neuroplasticity within the sympathetic nervous system but still induces a substantial decline in cardiac and hemodynamic function has not been forthcoming. To address this need we developed a moderately-severe midline contusion injury model at the T3 spinal level, measured in vivo hemodynamic and cardiac function invasively via catheterization, and quantified lesion site characteristics. Methods A total of 36 Wistar rats were assigned to either SCI (n=13) or naive (n=23) groups. For spinal cord contusion, animals were initially anesthetized with isoflurane then a dorsal midline incision was made and paraspinal musculature bluntly dissected. T3 laminectomy was performed and the animals were injured at the T3 spinal segment using an Infinite Horizon impactor with 300 kdyn of force. At 14 days post-SCI, left-ventricular and arterial catheterization was performed to assess in vivo cardiac and hemodynamic function. Following in vivo measures, the spinal cord was harvested, cut, and stained to determine lesion site characteristics via immunohistochemistry. Between-group differences in all outcomes were analyzed using an independent samples t-test. Statistical significance was set at p<0.05. Results SCI produced a decrease in left ventricular maximum pressure (LVmax) (naive=120±9 mmHg vs SCI=99±12 mmHg, p<0.001) and maximal left ventricular pressure rise (dP/dtmax) (9984±1049 vs. 6101±787 mmHg/s, p<0.001), arterial systolic blood pressure (127±14 vs. 97±10 mmHg, p<0.001), diastolic blood pressure (76±11 vs. 57±8 mmHg, p<0.001) and mean arterial pressure (93±12 vs. 69±8 mmHg, p<0.001). Immunohistological staining and visualization of the epicenter of the cord revealed 17±4% tissue sparing. Conclusions Our novel 300 kdyn contusion SCI produced significant decreases in cardiac and hemodynamic function while preserving 15-20% of tissue at the epicentre. Additionally, this injury model eased the burden of care compared to more severe injury models that are traditionally used in CV studies post-SCI. These findings suggest that our novel 300 kdyn contusion injury is a useful pre-clinical model of SCI to study rehabilitation approaches designed to induce neuroplasticity.

VX-765 reduces neuroinflammation after spinal cord injury in mice.

Inflammation is a major cause of neuronal injury after spinal cord injury. We hypothesized that inhibiting caspase-1 activation may reduce neuroinflammation after spinal cord injury, thus producing a protective effect in the injured spinal cord. A mouse model of T9 contusive spinal cord injury was established using an Infinite Horizon Impactor, and VX-765, a selective inhibitor of caspase-1, was administered for 7 successive days after spinal cord injury. The results showed that: (1) VX-765 inhibited spinal cord injury-induced caspase-1 activation and interleukin-1β and interleukin-18 secretion. (2) After spinal cord injury, an increase in M1 cells mainly came from local microglia rather than infiltrating macrophages. (3) Pro-inflammatory Th1Th17 cells were predominant in the Th subsets. VX-765 suppressed total macrophage infiltration, M1 macrophages/microglia, Th1 and Th1Th17 subset differentiation, and cytotoxic T cells activation; increased M2 microglia; and promoted Th2 and Treg differentiation. (4) VX-765 reduced the fibrotic area, promoted white matter myelination, alleviated motor neuron injury, and improved functional recovery. These findings suggest that VX-765 can reduce neuroinflammation and improve nerve function recovery after spinal cord injury by inhibiting caspase-1/interleukin-1β/interleukin-18. This may be a potential strategy for treating spinal cord injury. This study was approved by the Animal Care Ethics Committee of Bengbu Medical College (approval No. 2017-037) on February 23, 2017.

Complement C6 deficiency exacerbates pathophysiology after spinal cord injury

Historically, the membrane attack complex, composed of complement components C5b-9, has been connected to lytic cell death and implicated in secondary injury after a CNS insult. However, studies to date have utilized either non-littermate control rat models, or mouse models that lack significant C5b-9 activity. To investigate what role C5b-9 plays in spinal cord injury and recovery, we generated littermate PVG C6 wildtype and deficient rats and tested functional and histological recovery after moderate contusion injury using the Infinite Horizon Impactor. We compare the effect of C6 deficiency on recovery of locomotor function and histological injury parameters in PVG rats under two conditions: (1) animals maintained as separate C6 WT and C6-D homozygous colonies; and (2) establishment of a heterozygous colony to generate C6 WT and C6-D littermate controls. The results suggest that maintenance of separate homozygous colonies is inadequate for testing the effect of C6 deficiency on locomotor and histological recovery after SCI, and highlight the importance of using littermate controls in studies involving genetic manipulation of the complement cascade.

CRID3, a blocker of apoptosis associated speck like protein containing a card, ameliorates murine spinal cord injury by improving local immune microenvironment

BackgroundAfter spinal cord injury (SCI), destructive immune cell subsets are dominant in the local microenvironment, which are the important mechanism of injury. Studies have shown that inflammasomes play an important role in the inflammation following SCI, and apoptosis-associated speck-like protein containing a card (ASC) is the adaptor protein shared by inflammasomes. Therefore, we speculated that inhibiting ASC may improve the local microenvironment of injured spinal cord. Here, CRID3, a blocker of ASC oligomerization, was used to study its effect on the local microenvironment and the possible role in neuroprotection following SCI.MethodsMurine SCI model was created using an Infinite Horizon impactor at T9 vertebral level with a force of 50 kdynes and CRID3 (50 mg/kg) was intraperitoneally injected following injury. ASC and its downstream molecules in inflammasome signaling pathway were measured by western blot. The immune cell subsets were detected by immunohistofluorescence (IHF) and flow cytometry (FCM). The spinal cord fibrosis area, neuron survival, myelin preservation, and functional recovery were assessed.ResultsFollowing SCI, CRID3 administration inhibited inflammasome-related ASC and caspase-1, IL-1β, and IL-18 activation, which consequently suppressed M1 microglia, Th1 and Th1Th17 differentiation, and increased M2 microglia and Th2 differentiation. Accordingly, the improved histology and behavior have also been found.ConclusionsCRID3 may ameliorate murine SCI by inhibiting inflammasome activation, reducing proinflammatory factor production, restoring immune cell subset balance, and improving local immune microenvironment, and early administration may be a promising therapeutic strategy for SCI.

Role of Rho-associated coiled-coil containing protein kinase in the spinal cord injury induced neuropathic pain

BACKGROUND CONTEXTSpinal cord injury (SCI) can lead to increased phosphorylation of p38 in spinal cord microglia. This is one of the main causes for the development of persistent pain. Recently, we reported our study on the activation of p38 mitogen-activated protein kinases (MAPK) in spinal microglia, which has been considered the key molecule for the onset and maintenance of neuropathic pain after peripheral nerve injury, using a rat model. We also reported that the RhoA/Rho-associated coiled-coil containing protein kinase (ROCK) pathway mediates p38 activation in spinal microglia in peripheral nerve injury. But the precise mechanisms of neuropathic pain induced by SCI are still unclear. PURPOSEThis study aimed to examine the activation of microglia and the p38 MAPK expression in the lumbar spinal cord after thoracic SCI in rats, and the correlation to the therapeutic effect of ROCK inhibitor ripasudil in rats with SCI. STUDY DESIGNMale Sprague–Dawley rats underwent thoracic (T10) spinal cord contusion injury using an Infinite Horizon impactor device. SCI rats received ROCK inhibitor ripasudil (24 nmol/day or 240 nmol/day) from just before SCI to 3 days after SCI. METHODSThe mechanical threshold in the rat's hind paws was measured over four weeks. Morphology of microglia and phosphorylation of p38 (p-p38) in the lumbar spinal cord and were analyzed using immunohistochemistry. RESULTSThe p-p38 positive cell and Iba1 (a maker of microglia) positive area were significantly increased at the lumbar spinal dorsal horn (L4–5) 3 days and 7 days after SCI compared with the sham-control (p<.05), whereas phosphorylated p38 was co-localized with microglia. Three days after SCI, the intensity of phosphorylated p38 and Iba1 immunoreactive cells in the dorsal horn was significantly lower in the ripasudil treated groups than in the saline group. However, administration of ROCK inhibitor did not affect the numbers of microglia. Moreover, the withdrawal threshold of the ripasudil-treated rats was significantly higher than that of the saline-injected rats on 14 days and 28 days after SCI. CONCLUSIONSOur results suggest that activation of ROCK in spinal cord microglia is likely to have an important role in the activation of p38 MAPK, which has been considered as a key molecule that switches on neuropathic pain after SCI. Inhibition of ROCK signaling may offer a means in developing a novel neuropathic pain treatment after SCI. It may help patients with neuropathic pain after SCI. CLINICAL SIGNIFICANCEThe findings in the present study regarding intracellular mechanisms suggest that modulation of ROCK signaling may be a focus for novel treatment for neuropathic pain after SCI.

Relationship (?) Between Histological and Functional Measures in Moderate Contusion SCI in Adult Rat

To create a spinal cord injury (SCI), most laboratories implement a standardized method; however, considerable variability in injury size and associated functional deficits can occur across subjects. The present study was initiated to explore relationships between the histological features of spinal cord (SC) lesion and the associated functional deficits in locomotor, respiratory, and lower urinary tract (LUT) function in a widely used rat contusion SCI model that is generated using an Infinite Horizon impactor. To this end, adult female rats (n=8) received a moderate mid‐thoracic contusion SCI (200 kdyn, T8), and the impact displacement and force were recorded. Indices of functional recovery included: (1) BBB open field locomotor scoring at 1‐week post SCI, (2) total volume of expressed urine during the first 2‐weeks post SCI, and (3) diaphragm EMG activity and reflex micturition behavior and residual urine volume recorded during an acute cystometry experiment at 4‐weeks post SCI, after which the bladder was removed and weighed. Following fixation (4% PFA) and cryoprotection of the SC, 20 μm thick horizonal sections were obtained and counterstained with cresyl violet and Cyanine R / FeCl3 to visualize cell bodies and myelin, respectively, and the section at the level of the central canal was used as the site for preliminary analysis of the extent of lesion damage and degree of white matter sparing. The lesion area ranged from 27–49% and was measured by outlining the perimeter of the lesion site using a threshold tool and then normalized (%) to the area of a standardized 7 mm rostral‐caudal length region of SC that included the injury site. Myelin thickness was measured at 3 sites at the level of the injury and at the rostral and caudal ends of the tissue (uninjured) on each side of the SC, and the injury site measurements were normalized to the average myelin thickness of the corresponding uninjured side. A spared myelin index (%) using the larger minimum myelin thickness from the right or left side of the SC was then determined and ranged from 19–91%. For generation of the SCI, impactor displacement varied from 1287 to 1728 μm, with larger displacements being associated with larger injury sites and less myelin sparing. The lesion area, however, was not a strong predictor of myelin sparing, particularly in cases with asymmetrical lesions (n=2). Both lesion area and spared myelin index were reasonable predictors of post SCI recovery of locomotor (BBB scores) and LUT (expressed urine volume) function but not in predicting the degree of dysfunction in reflex micturition (increased residual volume) and bladder hypertrophy or inspiratory burst measures and their variability. These preliminary results suggest that factors in addition to the histological characteristics of the lesion site contribute to the degree of functional deficits/recovery following SCI.Support or Funding InformationDOD CDMRP W81XWH‐17‐1‐0260; NIH NS096514; NYS DOH SCIRB C32088GG

Treatment of rats with spinal cord injury using human bone marrow-derived stromal cells prepared by negative selection

BackgroundSpinal cord injury (SCI) is a highly debilitating pathology without curative treatment. One of the most promising disease modifying strategies consists in the implantation of stem cells to reduce inflammation and promote neural regeneration. In the present study we tested a new human bone marrow-derived stromal cell preparation (bmSC) as a therapy of SCI.MethodsSpinal cord contusion injury was induced in adult male rats at thoracic level T9/T10 using the Infinite Horizon impactor. One hour after lesion the animals were treated with a sub-occipital injection of human bmSC into the cisterna magna. No immune suppression was used. One dose of bmSC consisted, on average, of 2.3 million non-manipulated cells in 100 μL suspension, which was processed out of fresh human bone marrow from the iliac crest of healthy volunteers. Treatment efficacy was compared with intraperitoneal injections of methylprednisolone (MP) and saline. The recovery of motor functions was assessed during a surveillance period of nine weeks. Adverse events as well as general health, weight and urodynamic functions were monitored daily. After this time, the animals were perfused, and the spinal cord tissue was investigated histologically.ResultsRats treated with bmSC did not reject the human implants and showed no sign of sickness behavior or neuropathic pain. Compared to MP treatment, animals displayed better recovery of their SCI-induced motor deficits. There were no significant differences in the recovery of bladder control between groups. Histological analysis at ten weeks after SCI revealed no differences in tissue sparing and astrogliosis, however, bmSC treatment was accompanied with reduced axonal degeneration in the dorsal ascending fiber tracts, lower Iba1-immunoreactivity (IR) close to the lesion site and reduced apoptosis in the ventral grey matter. Neuroinflammation, as evidenced by CD68-IR, was significantly reduced in the MP-treated group.ConclusionsHuman bmSC that were prepared by negative selection without expansion in culture have neuroprotective properties after SCI. Given the effect size on motor function, implantation in the acute phase was not sufficient to induce spinal cord repair. Due to their immune modulatory properties, allogeneic implants of bmSC can be used in combinatorial therapies of SCI.

A transcriptomic study of probenecid on injured spinal cords in mice.

BackgroundRecent studies have found that probenecid has neuroprotective and reparative effects on central nervous system injuries. However, its effect on genome-wide transcription in acute spinal cord injury (SCI) remains unknown. In the present study, RNA sequencing (RNA-Seq) is used to analyze the effect of probenecid on the local expression of gene transcription 8 h after spinal injury.MethodsAn Infinite Horizon impactor was used to perform contusive SCI in mice. The SCI model was made by using a rod (1.3 mm diameter) with a force of 50 Kdynes. Sham-operated mice only received a laminectomy without contusive injury. The injured mice were randomly assigned into either the control (SCI_C) or probenecid injection (SCI_P) group. In the latter group, the probenecid drug was intraperitoneally injected (0.5 mg/kg) immediately following injury. Eight hours after the injury or laminectomy, the spinal cords were removed from the mice in both groups. The total RNAs were extracted and purified for library preparation and transcriptome sequencing. Differential gene expressions (DEGs) of the three groups—sham, SCI_C and SCI_P—were analyzed using a DESeq software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs were performed using a GOseq R package and KOBAS software. Real-time quantitative reverse-transcriptase polymerase chain reaction was used to validate RNA-Seq results.ResultsRNA-Seq showed that, compared to the SCI_C group, the number of DEGs was 641 in the SCI_P group (286 upregulated and 355 downregulated). According to GO analysis, DEGs were most enriched in extracellular matrix (ECM), collagen trimer, protein bounding and sequence specific DNA binding. KEGG analysis showed that the most enriched pathways included: cell adhesion molecules, Leukocyte transendothelial migration, ECM-receptor interactions, PI3K-Akt signaling pathways, hematopoietic cell lineages, focal adhesions, the Rap1 signaling pathway, etc. The sequence data have been deposited into the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/PRJNA554464).

Telemetric monitoring of penile pressure during mating in rats after chronic spinal cord injury.

In men with a spinal cord injury (SCI), erectile function, ejaculation, and fertility are severely impaired. The present study utilized a telemetric pressure transducer implanted into the corpus cavernosum of the penis to examine sexual function during awake mating behavior in a rat contusion model with a range of Infinite Horizon Impactor forces distributed between 150 and 215 kdyn. The mating behavior paradigm included examination of the counts, average pressure, and average duration for mounts, intromissions, and ejaculations. Male Wistar rats were mated with receptive females in 30-min sessions preinjury (sexual acclimation) and once per week for 6 wk beginning after a 2-wk recovery period post-SCI. All SCI animals had significant deficits in sexual function in the parameters measured. These deficiencies were more prevalent in a subset having less than 20% white matter sparing, likely a reflection of the extent of bilateral spino-bulbo-spinal sexual circuitry disruption at the lesion epicenter. The resulting discoordination of the autonomic and somatic reflex control of erection and ejaculation recorded using telemetry devices in an awake, behaving animal model provides an effective means of gauging sexual function deficits after SCI and could have utility for quantifying recovery after a therapeutic intervention.

Spatial and Cellular Expression Patterns of Erythropoietin-Receptor and Erythropoietin during a 42-Day Post-Lesional Time Course after Graded Thoracic Spinal Cord Impact Lesions in the Rat.

Erythropoietin (Epo) exhibits promising neuroregenerative potential for spinal cord injury (SCI), and might be involved in other long-term sequelae, such as neuropathic pain development. The current studies investigated the time courses and spatial and cellular patterns of Epo and erythropoietin receptor (EpoR) expression along the spinal axis after graded SCI. Male Long Evans rats received 100 kdyn, 150 kdyn, and 200 kdyn thoracic (T9) contusions from an Infinite Horizon impactor. Sham controls received laminectomies. Anatomical and quantitative immunohistochemical analyses of the EpoR/Epo expression along the whole spinal axis were performed 7, 15, and 42 postoperative days (DPO) after the lesioning. Cellular expression was investigated by double- and triple-labeling for EpoR/Epo with cellular markers and proliferating cells in subgroups of 5-bromo-2-deoxyuridine pre-treated animals. Prolonged EpoR/Epo-expression was confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Quantified EpoR/Epo immunoreactivities in pain-related spinal cord regions and ventrolateral white matter (VLWM) were correlated with the mechanical sensitivity thresholds and locomotor function of the respective animals. EpoR and Epo were constitutively expressed in the ventral horn neurons and vascular and glial cells in the dorsal columns (DC) and the VLWM. After SCI, in addition to expression in the lesion core, EpoR/Epo immunoreactivities exhibited significant time- and lesion grade-dependent induction in the DC and VLWM along the spinal axis. EpoR and Epo immunoreactive cells were co-stained with markers for astroglial, neural precursor cell and vascular markers. In the VLWM, EpoR- and Epo-positive proliferating cells were co-stained with glial fibrillary acidic protein (GFAP) and nestin. The DC EpoR/Epo immunoreactivities exhibited linear relationships with the behavioral correlates of post-lesional chronic pain development at DPO 42. SCI leads to long-lasting multicellular EpoR/Epo induction beyond the lesion core in the spinal cord regions that are involved in central pain development and regenerative processes. Our studies provide a time frame to investigate the effects of Epo application on motor function or pain development, especially in the later time course after lesioning.

The 3D characteristics of post-traumatic syringomyelia in a rat model: a propagation-based synchrotron radiation microtomography study.

Many published literature sources have described the histopathological characteristics of post-traumatic syringomyelia (PTS). However, three-dimensional (3D) visualization studies of PTS have been limited due to the lack of reliable 3D imaging techniques. In this study, the imaging efficiency of propagation-based synchrotron radiation microtomography (PB-SRµCT) was determined to detect the 3D morphology of the cavity and surrounding microvasculature network in a rat model of PTS. The rat model of PTS was established using the infinite horizon impactor to produce spinal cord injury (SCI), followed by a subarachnoid injection of kaolin to produce arachnoiditis. PB-SRµCT imaging and histological examination, as well as fluorescence staining, were conducted on the animals at the tenth week after SCI. The 3D morphology of the cystic cavity was vividly visualized using PB-SRµCT imaging. The quantitative parameters analyzed by PB-SRµCT, including the lesion and spared spinal cord tissue area, the minimum and maximum diameters in the cystic cavity, and cavity volume, were largely consistent with the results of the histological assessment. Moreover, the 3D morphology of the cavity and surrounding angioarchitecture could be simultaneously detected on the PB-SRµCT images. This study demonstrated that high-resolution PB-SRµCT could be used for the 3D visualization of trauma-induced spinal cord cavities and provides valuable quantitative data for cavity characterization. PB-SRµCT could be used as a reliable imaging technique and offers a novel platform for tracking cavity formation and morphological changes in an experimental animal model of PTS.

High-speed video analysis improves the accuracy of spinal cord compression measurement in a mouse contusion model

BackgroundAnimal models of spinal cord injuries aim to utilize controlled and reproducible conditions. However, a literature review reveals that mouse contusion studies using equivalent protocols may show large disparities in the observed impact force vs. cord compression relationship. The overall purpose of this study was to investigate possible sources of bias in these measurements. The specific objective was to improve spinal cord compression measurements using a video-based setup to detect the impactor-spinal cord time-to-contact. New methodA force-controlled 30kDyn unilateral contusion at C4 vertebral level was performed in six mice with the Infinite Horizon impactor (IH). High-speed video was used to determine the time-to-contact between the impactor tip and the spinal cord and to compute the related displacement of the tip into the tissue: the spinal cord compression and the compression ratio. Results & comparison with existing method(s)Delayed time-to-contact detection with the IH device led to an underestimation of the cord compression. Compression values indicated by the IH were 64% lower than those based on video analysis (0.33mm vs. 0.88mm). Consequently, the mean compression ratio derived from the device was underestimated when compared to the value derived from video analysis (22% vs. 61%). ConclusionsDefault time-to-contact detection from the IH led to significant errors in spinal cord compression assessment. Accordingly, this may explain some of the reported data discrepancies in the literature. The proposed setup could be implemented by users of contusion devices to improve the quantative description of the primary injury inflicted to the spinal cord.

Is There Additive Therapeutic Effect When GCSF Combined with Adipose-Derived Stem Cell in a Rat Model of Acute Spinal Cord Injury?

ObjectiveFunctional and neural tissue recovery has been reported in many animal studies conducted with stem cells. However, the combined effect of cytokines and stem cells has not yet been adequately researched. Here, we analyzed the additive effects of granulocyte colony-stimulating factor (GCSF) on adipose-derived stem cells (ADSCs) infusion in the treatment of acute spinal cord injury (SCI) in rats.MethodsFour days after intrathecal infusion tubes implantation in Sprague-Dawley rats, SCI was induced with an infinite horizon impactor. In the Sham group (n=5), phosphate-buffered saline was injected 3, 7, and 14 days after SCI. GCSF, ADSCs, and ADSCs with GCSF were injected at the same time in the GCSF (n=8), ADSC (n=8), and ADSC+GCSF groups (n=7), respectively.ResultsThe ADSC and ADSC+GCSF groups, but not the GCSF group, showed significantly higher Basso-Beattie-Bresnahan scores than the Sham group during 8 weeks (p<0.01), but no significant difference between the ADSC and ADSC+GCSF groups. In the ladder rung test, all four groups were significantly different from each other, with the ADSC+GCSF group showing the best improvement (p<0.01). On immunofluorescent staining (GAP43, MAP2), western blotting (GAP43), and reverse transcription polymerase chain reaction (GAP43, nerve growth factor), the ADSC and ADSC+GCSF groups showed higher levels than the Sham and GCSF groups.ConclusionOur analyses suggest that the combination of GCSF and ADSCs infusions in acute SCI in the rat does not have a significant additive effect. Hence, when combination agents for SCI stem cell therapy are considered, molecules other than GCSF, or modifications to the methodology, should be investigated.

Improvement in Lower Urinary Tract Function Following a Single Bout of Acute Intermittent Hypoxia in Rats with Chronic Spinal Cord Injury

Exposure to acute intermittent hypoxia (AIH) has been reported to improve respiratory and non‐respiratory somatic motor function in rodents and humans and has been proposed as a potential therapeutic intervention for treatment of chronic motor incomplete spinal cord injury (SCI). The goal of the present study is to characterize potential beneficial effects of AIH to improve lower urinary tract (LUT) function following incomplete SCI. To this end, female Sprague Dawley rats (225–250 g; n=11) received moderate midthoracic (T7–T9) spinal cord contusion injuries (200 kilodynes, Infinite Horizon Impactor; n=7) or sham injuries (spinal cord exposed, no injury; n=4) under isoflurane anesthesia. Following SCI, rats were inspected daily for SCI‐related issues (e.g., bladder/urinary tract infections, autophagia, overgrooming), and bladders were manually expressed until spontaneous voiding capability returned. Four weeks following the SCI/sham, bladder intravesical pressure and external urethral sphincter (EUS) EMG activity were recorded under urethane anesthesia (1.4 g/kg) during continuous infusion of saline (0.04 ml/min) into the bladder to elicit reflex micturition events (spontaneously breathing, vagus nerves intact). In addition, EMG data were acquired from the diaphragm and genioglossus muscles to assess effects on inspiratory motor outputs. Following 30–40 minutes of baseline recording, rats were exposed to a single bout of AIH consisting of three five‐minute episodes of hypoxia (10% O2; 90% N2) each separated by five‐minute exposures to room air. Following the AIH exposure, rats continued to spontaneously breathe room air, and data acquisition continued for up to 120 minutes. Prior to AIH, SCI rats exhibited pronounced rhythmic bladder activity consisting of both non‐voiding and voiding contractions. Typically, a series of non‐voiding bladder contractions with increasing amplitude preceded a voiding contraction, and the voiding contractions were incomplete resulting in continuous residual volume and an elevated minimum intravesical pressure. During each hypoxic episode during the AIH, the frequency of rhythmic bladder contraction increased and was associated with increased output volume and decreasing minimum intravesical pressure. In contrast, there was a marked decrease in rhythmic bladder activity during the normoxic period between hypoxic episodes with decreased output volume and increased minimum intravesical pressure. Immediately following the end of AIH, a similar decrease in rhythmic bladder contractions and output volume was observed and persisted for 5–10 minutes after which a more normal pattern of bladder activity (compared to baseline) emerged characterized by fewer non‐voiding contractions, increased output volume per volume per contraction, decreased minimum intravesical pressure, and a threshold‐driven pattern of bladder contraction that persisted up to 120 minutes post AIH. Sham operated rats exhibited a threshold‐driven pattern of bladder activity with efficient voiding and similar hypoxia‐dependent modulation of contraction frequency and increased output volume following AIH. These data suggest that AIH‐induced neuroplasticity results in improved LUT function in rats with chronic incomplete SCI and is a potentially promising therapeutic intervention for humans with SCI.Support or Funding InformationSupport for this project was provided by The Thomas Hartman Center for Parkinson's Research at Stony Brook University and The SUNY Brain Network of Excellence.

Neurogenic bladder dysfunction does not correlate with astrocyte and microglia activation produced by graded force in a contusion-induced spinal cord injury.

Rodent models for the study of neurogenic bladder dysfunction after spinal cord injury (SCI) are difficult to standardize, particularly when evaluating the specific contribution of the SCI to end-organ function. The purpose of this study was to evaluate the degree of bladder dysfunction associated with a highly reproducible, contusion-induced SCI in female rats. An infinite horizon impactor was used to create a contusion SCI with a magnitude of either 100 or 150 kDyne at the T8/T9 thoracic region of female Sprague-Dawley rats. Locomotor function, and the presence of astrocytes (positive regions for Glial Fibrillary Acidic Protein) and microglia (positive cells for the integrin CD11b) at the SCI site were determined at four weeks after SCI. Similarly, cystometric properties were characterized in urethane anesthetized rats at four weeks post-SCI. The significant increases in astrocyte and microglia in the T8/T9 region in all of the SCI animals did not correlate with locomotor impairment or bladder dysfunction. After performing the cystometric studies substantial differences were found in both SCI groups when compared to intact animals, specifically a high frequency of non-voiding contractions, different durations for intraluminal pressure-high frequency oscillations, intercontractile intervals, impaired micturition volumes, and estimated voiding efficiency. These results suggest that a contusion SCI can increase microglia and astrocyte activation without a strong association with bladder dysfunction. The present study will be important for precise considerations about correlating the intensity of an SCI with impairment outcomes at both locomotor or organ function levels.