Matrix Metalloproteinase-9 is Involved in the Fibrotic Process in Denervated Muscles after Sciatic Nerve Trauma and Recovery.

Fibrosis of the injured muscles is a problem of recovery from trauma and denervation. The aim of the work was to investigate the interconnection of matrix metalloproteinase-9 (ММР-9) activity in denervated muscles with fibrosis and to estimate its role in nerve restoration by the epineurial suture, fibrin-based glue, and polyethylene glycol hydrogel. The activity of matrix metalloproteinases was estimated by gelatin zymography. Collagen density in muscles was determined histochemically. An increased level of the active MMP-9 is associated with the fibrous changes in the denervated skeletal muscles and after an epineurial suture. The use of fibrin glue and polyethylene glycol hydrogel resulted in a lower level of collagen and ММР-9 activity, which may be a therapeutic target in the treatment of neuromuscular lesions, and has value in fibrosis analysis following microsurgical intervention for peripheral nerve reconstruction.

Regulation of the apoptosis/autophagy switch by propionic acid in ventromedial hypothalamus of rats with type 2 diabetes mellitus.

Background: Hypothalamic dysregulation may cause abnormal glucose metabolism and type 2 diabetes mellitus (T2DM). The balance between autophagy and apoptosis is important for maintaining cellular/tissue homeostasis and may be disrupted in T2DM. Objectives: Since propionic acid (PA) exerts neuroprotective effects, the aim was to investigate its effects on apoptosis/autophagy switch in the ventromedial hypothalamus (VMH) of T2DM rats. Materials and methods: Male Wistar rats were divided: 1) control; 2) T2DM; groups that received (14 days, orally): 3) metformin (60 mg/kg); 4) sodium salt of PA (60 mg/kg); 5) PA + metformin. Western blotting (Bax, Bcl-xl, LC3, Beclin-1, caspase-3), RT-PCR (Bax, Bcl-xl, LC3, Beclin-1), transmission electron microscopy and immunohistochemical staining (Bax, Bcl-xl) were performed on the VMH samples. Results: T2DM-induced apoptosis and mitoptosis, enlarged endoplasmic reticulum (ER) tubules/cisterns were observed in VMH, and accompanied by an imbalance of pro- and anti-apoptotic factors: elevation of pro-apoptotic markers Bax and caspase-3, decrease in autophagy protein LC3 and anti-apoptotic Bcl-xl. Metformin and PA administration partially improved VMH ultrastructural changes by reducing mitochondrial swelling and diminishing the number of apoptotic neurons. Metformin inhibited neuronal apoptosis, however, caused reactive astrogliosis and accumulation of lipofuscin granules. Elevated number of autophagosomes was associated with the LC3, Beclin-1 and Bcl-xl increase and decrease in Bax and caspase-3 vs. T2DM. PA switched cell fate from apoptosis to autophagy by elevating LC3 and Beclin-1 levels, increasing Bcl-xl content that altogether may represent adaptive response to T2DM-induced apoptosis. PA + metformin administration lowered relative area of ER membranes/cisterns vs. control, T2DM and metformin, and was optimal considering ratio between the pro-apoptotic, anti-apoptotic and autophagy markers. Conclusion: T2DM was associated with apoptosis activation leading to impairments in VMH. PA in combination with metformin may be effective against diabetes-induced cell death by switching apoptosis to autophagy in VMH.

Effect of Propionic Acid on Diabetes-Induced Impairment of Unfolded Protein Response Signaling and Astrocyte/Microglia Crosstalk in Rat Ventromedial Nucleus of the Hypothalamus.

Background: The aim was to investigate the influence of propionic acid (PA) on the endoplasmic reticulum (ER), unfolded protein response (UPR) state, and astrocyte/microglia markers in rat ventromedial hypothalamus (VMH) after type 2 diabetes mellitus (T2DM). Methods: Male Wistar rats were divided: (1) control, (2) T2DM, and groups that received the following (14 days, orally): (3) metformin (60 mg/kg), (4) PA (60 mg/kg), and (5) PA+metformin. Western blotting, RT-PCR, transmission electron microscopy, and immunohistochemical staining were performed. Results: We found T2DM-associated enlargement of ER cisterns, while drug administration slightly improved VMH ultrastructural signs of damage. GRP78 level was 2.1-fold lower in T2DM vs. control. Metformin restored GRP78 to control, while PA increased it by 2.56-fold and metformin+PA-by 3.28-fold vs. T2DM. PERK was elevated by 3.61-fold in T2DM, after metformin-by 4.98-fold, PA-5.64-fold, and metformin+PA-3.01-fold vs. control. A 2.45-fold increase in ATF6 was observed in T2DM. Metformin decreased ATF6 content vs. T2DM. Interestingly, PA exerted a more pronounced lowering effect on ATF6, while combined treatment restored ATF6 to control. IRE1 increased in T2DM (2.4-fold), metformin (1.99-fold), and PA (1.45-fold) groups vs. control, while metformin+PA fully normalized its content. The Iba1 level was upregulated in T2DM (5.44-fold) and metformin groups (6.88-fold). Despite PA treatment leading to a further 8.9-fold Iba1 elevation, PA+metformin caused the Iba1 decline vs. metformin and PA treatment. GFAP level did not change in T2DM but rose in metformin and PA groups vs. control. PA+metformin administration diminished GFAP vs. PA. T2DM-induced changes were associated with dramatically decreased ZO-1 levels, while PA treatment increased it almost to control values. Conclusions: T2DM-induced UPR imbalance, activation of microglia, and impairments in cell integrity may trigger VMH dysfunction. Drug administration slightly improved ultrastructural changes in VMH, normalized UPR, and caused an astrocyte activation. PA and metformin exerted beneficial effects for counteracting diabetes-induced ER stress in VMH.

Experimental unilateral intracerebral hemorrhage induces delayed bilateral neurodegeneration of sciatic nerve fibres in rats.

Pathological processes, such as inflammatory effects, oxidative stress, apoptosis and cytotoxicity of blood after an intracerebral hemorrhage (ICH), generally contribute to a secondary injury. One of the secondary ICH consequences in the nervous system may be delayed neurodegeneration of the peripheral nerves. Therefore, the aim of our study was to investigate possible structural changes in the sciatic nerve and changes in the conduction velocity via this nerve at different terms after experimental ICH in male Wistar rats. Intracerebral hemorrhage was provided by direct injection of autologous blood into the capsula interna. On the 10th day after ICH mean conduction velocity in sciatic nerve was 15% smaller compared to the control. On the 30th and 90th days after ICH, highly significant decreases in the conduction velocity by 62% and 60%, respectively in comparison with the control group of animals were observed. The data of morphometric analysis demonstrated significant decreases in the mean diameter and density of myelinated fibres at all examined terms after ICH. A number of the myelin sheaths were swollen and lost their regular laminations. Axoplasmic and myelin degenerations were the most frequent events in these nerve fibres; reductions of the diameter of the axis cylinders were also observed. In the contralateral nerve (related to the hemisphere with ICH), negative changes were greater, while the ipsilateral nerve was also subjected to those. Our data demonstrate that the consequences of unilateral ICH in the capsula interna induce bilateral negative changes in the peripheral nervous system of rats.

CARDIOVASCULAR SYSTEM OF THE MATURE RATS WITH CONGENITAL HYPOTHYROIDISM AND ARTERIAL HYPERTENSION.

OBJECTIVE: The aim: Studying changes in the ultrastructure of blood circulatory capillaries of the myocardium of mature rats with hypothyroidism and arterial hypertension. PATIENTS AND METHODS: Materials and methods: Experiments were conducted on (240 days) 10 ISIAH (inherited stress-induced arterial hypertension) line rats with AH (arterial hypertension), 10 Wistar line rats with congenital hypothyroidism and 10 intact animals. Arterial pressure was measured, and the development of hypothyroidism was controlled by the immune enzyme method. The study of the left ventricle myocardium of the rat heart was carried out by electron microscopic and morphometric studies. RESULTS: Results: In in rats with AH the following changes were observed in the blood capillaries of the myocardium: decrease in the number of capillaries; disturbance of blood circulation; the number of organelles of the biosynthetic plan and structures involved in the transendothelial transfer of substances decreased in endothelial cells; lysis and edema of the latter; mucinous perivascular edema, confirmed by the accumulation of fine-fibrillar structures, collagen fibers, cellular detritus. By the same term, in the group with congenital hypothyroidism, dystrophic-destructive changes in the blood capillaries of the myocardium acquired the highest degree, which resulted in a decrease in their number due to destruction. Ultrastructure of the biosynthetic plan organelles and structures of the transendothelial transfer of substances were in decompensated state. CONCLUSION: Conclusions: The rats (in 240 days) with AH and congenital hypothyroidism express breakdown of compensatory processes in the capillaries of the myocardium. This is manifested by the further dilution of capillaries, the development of hypoxic state in them as well as mucinous edema of interstitium, the decrease of activity of biosynthetic and transport processes.

Peripheral nerve gap management: rock-paper-scissors

Peripheral nerves injury is one of the topical medical, social and economic problems. One of the crucial factors of surgical treatment of nerve trunks injuries ensuring their successful regeneration is the precise and tight connection of the proximal and distal stumps. To fulfill this goal a whole wealth of suturing and adhesive materials, laser and high-frequency electric welding techniques were suggested. Currently microsurgical autoneurografting, which is considered the gold standard, is preferred in repairing nerve trunks defects. However, although effective, this surgical intervention has certain limitations, including the injury at the site of donor nerve harvesting with subsequent hypo­ or anesthesia, scarring of the donor site, instances of formation of the painful neuroma of the central stump of the cutaneous nerve, lack of grafting material in the case of significant defect of the injured nerve or during reoperation, mismatch between the bundle structure of the damaged nerve trunk and the grafted segments of the cutaneous nerve. This situation stimulates the search for alternatives to autoneurografting.

Nerve Regeneration in Conditions of HSV-Infection and an Antiviral Drug Influence.

Herpes simplex virus type I (HSV-I) is a latent neuroinfection which can cause focal brain lesion. The role of HSV-infection in nerve regeneration has not been studied so far. The aim of the work was to study sciatic nerve regeneration in the presence of HSV-infection and the influence of an antiviral drug. BALB/c line mice were divided into five groups. Group 1 animals were infected with HSV-I. After resolution of neuroinfection manifestations the sciatic nerve of these animals was crushed. Group 2 mice were administered acyclovir following the same procedures. Groups 3-5 mice served as controls. Thirty days after the operation distal nerve stumps and m.gastrocnemius were studied morphologically and biochemically. Ultrastructural organization of the sciatic nerve in control animals remained intact. Morphometric parameters of the nerves from the experimental groups have not reach control values. However, in the group 1 diameter of nerve fibers was significantly smaller than in the group 2. Both nerve regeneration and m.gastrocnemius reinnervation were confirmed. The muscle hypotrophy was found in groups 1, 2, and 3 (the muscle fibers diameter decreased). Metabolic changes in the muscles of the infected animals (groups 1 and 2) were more pronounced than in control groups 3 and 4. The levels of TBA-active products, conjugated dienes, carbonyl and SH-groups were reduced in m.gastrocnemius of the experimental groups, however no significant difference associated with acyclovir administration was found. HSV-infection is not limited to the local neurodegenerative changes in the CNS but affects regeneration of the injured sciatic nerve. Anat Rec, 301:1734-1744, 2018. © 2018 Wiley Periodicals, Inc.

The influence of lead nanoparticles on the morpho-functional changes of rat liver during the postexposure period.

Lead as any heavy metals may be found in soil, water, air, and is used in everyday life. Once in the body, it causes toxic effect, making the liver, which is one of the main organs of detoxification, suffer. Recently, the study of the action of not only ionic forms of lead, but also its nanoparticles, has become topical. The study aims at determining changes in the liver of rats and biochemical changes in their blood both at late term of exposure to nanoparticles of lead compounds and in the post-exposure period. The study was performed on 120 male rats of Wistar line, which were divided into two series, each series containing four groups. The first and the second groups of animals were intraperitoneally injected with colloidal solution of nanoparticles of lead sulfide of 10 and 30 nm in size, and the third group were intraperitoneally injected with a solution of lead nitrate. The fourth group of animals served as control. In the first series, the investigated substances were administered 60 times within 12 weeks. In the second series, after 60-fold administration of the investigated substances, the exposure was discontibued and animals were observed for 6 weeks-overall duration of 18 weeks. Histological, morphometrical and biochemical methods were used. The body weight was reduced in the rats exposed to PbSnano1 at week 12 of experiment and in rats exposed to both PbSnano1 and Pb(NO3 )2 in the second series. Absolute liver weight increased at week 12 of experiment in all experimental groups. In the second series this value almost reached that of the control level. Relative liver weight in the animals of all experimental groups was higher than that in the control at week 12 of experiment. In the second series this value remained higher in rats exposed to PbSnano1 . After 12 weeks of exposure dystrophic changes in the liver were found in all experimental groups. At week 6 after the exposure (the second series) destructive changes in the liver decreased. Total protein, albumin, glucose, total lipids, cholesterol, triglycerides content in blood serum corresponded with morphological data. The experiment has demonstrated that the 12 weeks long exposure to lead nanoparticles had harmful effect on the liver. Within the postexposure 6-weeks period structural changes in the liver and biochemical changes in blood serum decreased. Biochemical changes in blood serum corresponded to the morphological data. By many parameters PbSnano1 had more pronounced harmful effect. Toxicity of PbSnano2 and Pb(NO3 )2 were comparable.

New Method of Injured Nerve Repair.

BACKGROUND: Innovative surgical techniques form the basis of therapeutic approaches to address the negative consequences of nerve damage. This study evaluated the effectiveness of nerve trunk regeneration after the use of an electrosurgical instrument by looking at the patterns of morphological changes in the injured nerve and the structural elements of the segment motor center. METHODOLOGY: The study was performed on male Wistar rats divided into four groups: group 1, control; group 2, rats with simulated sciatic nerve injury with epineural sutures; 3, rats subjected to an experimental surgical procedure using high-frequency electric welding technology; and 4, rats with simulated sciatic nerve injury without posttransection repair. To study changes in the peripheral stump of the transected nerves and L5 segments of the spinal cord, we used histologic, immunohistochemical, and morphometric methods. RESULTS: At week 12 after the surgery, there were more S-100+ Schwann cells, increased expression of neurofilaments (NFs), and glial fibrillary acidic protein in the peripheral stump in group 3 than in groups 2 and 4, which indicates enhanced neurotization and myelination. Group 3 animals demonstrated reduced expression of S-100 and NFs in the motor center of the spinal cord compared with group 2 that suggests less pronounced reactive changes caused by electric welding technology. CONCLUSIONS: The study showed a novel surgical method using an electrosurgical instrument in a welding mode to stimulate regeneration of the injured nerve and to cause less prominent reactive changes in its segment motor center.