Histomorphometric study of the soleus muscle under conditions of modeling of spinal cord contusion injury: experimental morphological study

Objective. To conduct a morphometric analysis of the soleus muscle of rats after moderate spinal cord contusion injury.Material and Methods. Experiments were performed on female Wistar rats aged 8–12 months, weighing 270–320 g. Animals of the experimental group (n = 25) underwent laminectomy at the T9 level under general anesthesia and modeling of spinal contusion injury of moderate severity. Intact rats constituted the control group (n = 10). Euthanasia was performed on the 5th, 15th, 30th, 60th, 90th, and 180th days of the experiment. Paraffin sections were stained with hematoxylin-eosin and Masson, the diameters of muscle fibers were determined by computer morphometry, and histograms of their distribution were obtained.Results. In the soleus muscle, the signs of reversible reparative processes prevailed in response to neurotrophic damage. It was evidenced by a local increase in the diversity of myocyte diameters and the loss of polygonality of their profiles, focal destruction of muscle fibers, activation of the connective tissue component, disorganization of some intramuscular nerve conductors, and vascular fibrosis of perimysium. Nevertheless, the histostructure of an intact muscle prevailed in the course of the experiment, which was confirmed by the data of morphometric analysis. All histograms of the distribution of the muscle fiber diameters are unimodal with a mode in the range of 30–41 μm. On the 180th day, the maximum myocyte diameters in the histogram of the left limb muscle belonged to the range of 21–30 μm, which was typical for histograms in the intact group.Conclusion. The nature of the plastic reorganization of the soleus muscle when neurotrophic control is impaired indicates compensatory regeneration of muscle tissue by the type of restitution, which opens up the possibility of predicting the rehabilitation period. It is advisable to take this into account when developing medical and social programs and therapeutic measures, where the most important role is played by superficial neuromuscular and functional electrical stimulation.

Limited changes in locomotor recovery and unaffected white matter sparing after spinal cord contusion at different times of day

The circadian gene expression rhythmicity drives diurnal oscillations of physiological processes that may determine the injury response. While outcomes of various acute injuries are affected by the time of day at which the original insult occurred, such influences on recovery after spinal cord injury (SCI) are unknown. We report that mice receiving moderate, T9 contusive SCI at ZT0 (zeitgeber time 0, time of lights on) and ZT12 (time of lights off) showed similar hindlimb function recovery in the Basso mouse scale (BMS) over a 6 week post-injury period. In an independent study, no significant differences in BMS were observed after SCI at ZT18 vs. ZT6. However, the ladder walking test revealed modestly improved performance for ZT18 vs. ZT6 mice at week 6 after injury. Consistent with those minor effects on functional recovery, terminal histological analysis revealed no significant differences in white matter sparing at the injury epicenter. Likewise, blood-spinal cord barrier disruption and neuroinflammation appeared similar when analyzed at 1 week post injury at ZT6 or ZT18. Therefore, locomotor recovery after thoracic contusive SCI is not substantively modulated by the time of day at which the neurotrauma occurred.

Deletion or Inhibition of Astrocytic Transglutaminase 2 Promotes Functional Recovery after Spinal Cord Injury

Following CNS injury, astrocytes become “reactive” and exhibit pro-regenerative or harmful properties. However, the molecular mechanisms that cause astrocytes to adopt either phenotype are not well understood. Transglutaminase 2 (TG2) plays a key role in regulating the response of astrocytes to insults. Here, we used mice in which TG2 was specifically deleted in astrocytes (Gfap-Cre+/− TG2fl/fl, referred to here as TG2-A-cKO) in a spinal cord contusion injury (SCI) model. Deletion of TG2 from astrocytes resulted in a significant improvement in motor function following SCI. GFAP and NG2 immunoreactivity, as well as number of SOX9 positive cells, were significantly reduced in TG2-A-cKO mice. RNA-seq analysis of spinal cords from TG2-A-cKO and control mice 3 days post-injury identified thirty-seven differentially expressed genes, all of which were increased in TG2-A-cKO mice. Pathway analysis revealed a prevalence for fatty acid metabolism, lipid storage and energy pathways, which play essential roles in neuron–astrocyte metabolic coupling. Excitingly, treatment of wild type mice with the selective TG2 inhibitor VA4 significantly improved functional recovery after SCI, similar to what was observed using the genetic model. These findings indicate the use of TG2 inhibitors as a novel strategy for the treatment of SCI and other CNS injuries.

Deletion or inhibition of astrocytic transglutaminase 2 promotes functional recovery after spinal cord injury

Following CNS injury astrocytes become “reactive” and exhibit pro-regenerative or harmful properties. However, the molecular mechanisms that cause astrocytes to adopt either phenotype are not well understood. Transglutaminase 2 (TG2) plays a key role in regulating the response of astrocytes to insults. Here we used mice in which TG2 was specifically deleted in astrocytes (Gfap-Cre+/-TG2fl/fl, referred to here as TG2-A-cKO) in a spinal cord contusion injury (SCI) model. Deletion of TG2 from astrocytes resulted in a significant improvement in motor function following SCI. GFAP and NG2 immunoreactivity, as well as number of SOX9 positive cells, were significantly reduced in TG2-A-cKO_mice. RNA-seq analysis of spinal cords from TG2-A-cKO and control mice 3 days postinjury identified thirty-seven differentially expressed genes, all of which were increased in TG2-A-cKO mice. Pathway analysis reveals a prevalence for fatty acid metabolism, lipid storage and energy pathways, which play essential roles in neuron-astrocyte metabolic coupling. Excitingly, treatment of wild type mice with the selective TG2 inhibitor VA4 significantly improved functional recovery after SCI, similar to what was observed using the genetic model. These findings indicate the use of TG2 inhibitors as a novel strategy for the treatment of SCI and other CNS injuries.