Thiago Luiz Russo

ELECTRICAL STIMULATION IMPAIRS EARLY FUNCTIONAL RECOVERY AND ACCENTUATES SKELETAL MUSCLE ATROPHY AFTER SCIATIC NERVE CRUSH INJURY IN RATS

Neuromuscular recovery after peripheral nerve lesion depends on the regeneration of severed axons that re‐establish their functional connection with the denervated muscle. The aim of this study was to determine the effects of electrical stimulation (ES) on the neuromuscular recovery after nerve crush injury in rats. Electrical stimulation was carried out on the tibialis anterior (TA) muscle after sciatic nerve crush injury in a rat model. Six ES sessions were administered every other day starting from day 3 postinjury until the end of the experiment (day 14). The sciatic functional index was calculated. Muscle excitability, neural cell adhesion molecule (N‐CAM) expression, and muscle fiber cross‐sectional area (CSA) were accessed from TA muscle. Regenerated sciatic nerves were analyzed by light and confocal microscopy. Both treated (crush+ES) and untreated (crush) groups had their muscle weight and CSA decreased compared with the normal group (P < 0.05). Electrical stimulation accentuated muscle fiber atrophy more in the crush+ES than in the crush group (P < 0.05). N‐CAM expression increased in both crush and crush+ES groups compared with the normal group (P < 0.05). Regenerated nerves revealed no difference between the crush and crush+ES groups. Nevertheless, functional recovery at day 14 post‐injury was significantly lower in crush+ES group compared with the crush group. In addition, the crush+ES group had chronaxie values significantly higher on days 7 and 13 compared with the crush group, which indicates a decrease in muscle excitability in the crush+ES animals. The results of this study do not support a benefit of the tested protocol of ES during the period of motor nerve recovery following injury. Muscle Nerve, 2010

Effects of 660 and 780 nm low‐level laser therapy on neuromuscular recovery after crush injury in rat sciatic nerve

Post‐traumatic nerve repair is still a challenge for rehabilitation. It is particularly important to develop clinical protocols to enhance nerve regeneration. The present study investigated the effects of 660 and 780 nm low‐level laser therapy (LLLT) using different energy densities (10, 60, and 120 J/cm2) on neuromuscular and functional recovery as well as on matrix metalloproteinase (MMP) activity after crush injury in rat sciatic nerve.

Electrical stimulation based on chronaxie reduces atrogin-1 and myoD gene expressions in denervated rat muscle.

Denervation induces muscle fiber atrophy and changes in the gene expression rates of skeletal muscle. Electrical stimulation (ES) is a procedure generally used to treat denervated muscles in humans. This study evaluated the effect of ES based on chronaxie and rheobase on the expression of the myoD and atrogin‐1 genes in denervated tibialis anterior (TA) muscle of Wistar rats. Five groups were examined: (1) denervated (D); (2) D+ES; (3) sham denervation; (4) normal (N); and (5) N+ES. Twenty muscle contractions were stimulated every 48 h using surface electrodes. After 28 days, ES significantly decreased the expression of myoD and atrogin‐1 in D+ES compared to the D group. However, ES did not prevent muscle‐fiber atrophy after denervation. Thus, ES based on chronaxie values and applied to denervated muscles using surface electrodes, as normally used in human rehabilitation, was able to reduce the myoD and atrogin‐1 gene expressions, which are related to muscular growth and atrophy, respectively. The results of this study provide new information for the treatment of denervated skeletal muscle using surface ES. Muscle Nerve, 2006

Electrical stimulation increases matrix metalloproteinase‐2 gene expression but does not change its activity in denervated rat muscle

Muscle‐fiber atrophy occurs concomitantly with intramuscular connective tissue proliferation following denervation. These events contribute to the impairment of mechanical and functional properties of denervated muscles and compromise their recovery. Electrical stimulation (ES) is used in human rehabilitation to treat denervated muscles. However, the effects of this therapy on the intramuscular extracellular matrix (ECM) remain uncertain. Metalloproteinases (MMPs) are responsible by remodeling ECM in many neuromuscular disorders. This study evaluates the effect of ES on the activity of two important MMPs, MMP‐2 and MMP‐9, both involved in ECM remodeling of rat denervated muscles. Thirty‐four Wistar rats (3 months old, 356 ± 38.7 g) were divided into five groups: denervated (D); D+ES; sham denervation; normal (N); and N+ES. Twenty maximal muscle contractions were stimulated every 48 h using surface electrodes, as generally used in the rehabilitation of human denervated muscle. Both zymographic analysis and real‐time polymerase chain reaction (PCR) of MMPs were used to evaluate muscle after denervation for 28 days. Both the D and D+ES groups showed increased MMP‐2 activity compared with the N group (P < 0.05). Furthermore, only the D+ES had increased MMP‐2 gene expression compared with the N group (P < 0.05). MMP‐9 activity was not detected in any of the groups. The results of this study indicate that denervation increases MMP‐2 activity, and ES regulates MMP‐2 gene expression in rat denervated skeletal muscle. These findings clarify the effects of ES on the ECM of denervated muscle and may be helpful in designing new therapeutic strategies for rehabilitation in patients with denervation of muscle. Muscle Nerve, 2008

Effect of Low-Level Laser Therapy (LLLT) on Acute Neural Recovery and Inflammation-Related Gene Expression After Crush Injury in Rat Sciatic Nerve

Peripheral nerve function can be debilitated by different kinds of injury. Low‐level laser therapy (LLLT) has been used successfully during rehabilitation to stimulate recovery. The aim of this study was to evaluate the effects of LLLT (660 nm, 60 J/cm2, 40 mW/cm2) on acute sciatic nerve injury.

Joint inflammation alters gene and protein expression and leads to atrophy in the tibialis anterior muscle in rats.

Objective: The aim of this study was to evaluate the effect of tibiotarsal joint inflammation in rat tibialis anterior muscle through muscle fiber cross-sectional area (CSA) and gene expression (atrogin-1, muscle ring finger-1 [MuRF1], myogenic differentiation-1 [MyoD], p38 mitogen-activated protein kinase [p38MAPK], nuclear factor kappa B-dependent [NF&kgr;B], tumor necrosis factor-alpha [TNF-&agr;]). Design: Wistar rats were randomly divided into three periods (2, 7, and 15 days) and assigned into four groups within each experimental period: control, sham, inflammation, and immobilization. Real-time polymerase chain reaction, Western blot, immunofluorescence, and muscle fiber CSA analyses were performed. Results: At 2 days, the inflammation group increased atrogin-1, MuRF1, and myostatin and reduced MyoD expression. At 7 days, the inflammation group increased atrogin-1, MuRF1, NF&kgr;B, p38MAPK, MyoD, myostatin, and TNF-&agr; expression and TNF-&agr; protein and reduced muscle fiber CSA. At 15 days, gene and protein expression in the inflammation group returned to basal levels, and CSA values were similar to those in control and sham groups. The immobilization groups have a similar pattern in all experimental periods, with increased atrogin-1, MuRF1, NF&kgr;B, and TNF-&agr; gene expression and reduced muscle fiber CSA. The sham group had increased myostatin and atrogin-1 expression at 2 days and increased MyoD and myostatin expression at 7 days. Conclusions: Joint inflammation stimulated the expression of muscle factors related to atrophy, growth, differentiation, and mass regulation followed by muscle atrophy.

Stretching and electrical stimulation regulate the metalloproteinase-2 in rat denervated skeletal muscle

Abstract Objective: To evaluate the effects of electrical stimulation (ES), stretching and their combined effects in matrix metalloproteinases (MMPs) gene expression and activity during early denervation of rat tibialis anterior (TA) muscle by axonotmesis. Methods: Thirty adult male Wistar rats were divided into five groups: normal (N), denervated TA (D), denervated TA submitted to daily ES (D + ES), denervated TA submitted to daily stretching (D + St) and denervated TA submitted daily to both ES and stretching concurrently (D + ES + St). Both zimographic analysis and real time polymerase chain reaction of MMPs were used to muscular evaluation. In addition, muscle fiber cross-section area (CSA) was also evaluated. Results: Stretching increased MMP-2 activity in denervated muscle when performed alone as well as in association with ES (p<0·01). Both stretching and ES, individually and in association, increased MMP-2 gene expression in denervated muscle compared to N and D (p<0·05). All denervated groups decreased muscle fiber CSA compared to N (p<0·05). Discussion: While stretching is the main stimulus leading to the activation of MMP-2, both ES and stretching are able to increase MMP-2 gene expression in rat denervated muscle suggesting ECM remodeling.

Effects of electrical stimulation and stretching on the adaptation of denervated skeletal muscle: implications for physical therapy

BACKGROUND This review will describe the main cellular mechanisms involved in the reduction and increase of myoproteins synthesis commonly associated with muscle atrophy and hypertrophy, respectively. OBJECTIVE We analyzed the effects of electrical stimulation (ES) and stretching exercise on the molecular pathways involved in muscle atrophy and hypertrophy. We also described the main effects and limits of these resources in the skeletal muscle, particularly on the denervated muscle. DISCUSSION Recently, our studies showed that the ES applied in a similar manner as performed in clinical practice is able to attenuate the increase of genes expression involved in muscle atrophy. However, ES was not effective to prevent the loss of muscle mass caused by denervation. Regarding to stretching exercises, their mechanisms of action on the denervated muscle are not fully understood and studies on this area are scarce. Studies from our laboratory have found that stretching exercise increased the extracellular matrix remodeling and decreased genes expression related to atrophy in denervated muscle. Nevertheless, it was not enough to prevent muscle atrophy after denervation. CONCLUSIONS In spite of the use of stretching exercise and ES in clinical practice in order to minimize the atrophy of denervated muscle, there is still lack of scientific evidence to justify the effectiveness of these resources to prevent muscle atrophy in denervated muscle.

Muscle and Nerve Responses After Different Intervals of Electrical Stimulation Sessions on Denervated Rat Muscle

Lima SC, Caierão QM, Peviani SM, Russo TL, Somazz MC, Salvini TF, Teodori RM, Minamoto VB: Muscle and nerve responses after different intervals of electrical stimulation sessions on denervated rat muscle. Objective:Electrical stimulation is a procedure used to treat denervated muscles. The number of electrical stimulation sessions varies across muscle rehabilitation protocols, from daily to certain days throughout the week. The purpose of this study was to evaluate how muscle and nerve respond to different intervals of electrical stimulation applied to denervated muscle. Design:Denervation of rat gastrocnemius muscle was imposed via nerve crush, and electrical stimulation was applied to the muscle either daily (Monday through Friday) or on alternate days (Monday, Wednesday, and Friday). Four experimental groups were studied: denervated, denervated plus daily electrical stimulation, denervated plus alternate-day electrical stimulation, and control. Results:On the 12th day after nerve crush, levels of MyoD, myostatin, and atrogin-1 gene expression, as well as muscle fiber and nerve morphometry, were evaluated. Expression levels of all three genes were higher in the denervated group when compared with control. Also, expression levels of MyoD and myostatin were higher in denervated plus alternate-day electrical stimulation and denervated plus daily electrical stimulation groups when compared with denervated. The denervated plus daily electrical stimulation group had lower atrogin-1 expression, lower density of intramuscular connective tissue, and better morphometric nerve characteristics when compared with the denervated and denervated plus alternate-day electrical stimulation. Conclusions:These results indicate that the responses of both muscle and nerve to electrical stimulation after muscle denervation depend on the intervals of electrical stimulation application.

Effects of alternagin-C from Bothrops alternatus on gene expression and activity of metalloproteinases in regenerating skeletal muscle

This study evaluated the effects of alternagin-C (ALT-C) on mRNA levels of VEGF, MyoD and matrix metalloproteinase 2 (MMP-2) and on activity of MMPs in injured tibialis anterior (TA) muscle induced by cryolesioning in rats. Thirty-six Wistar rats (3 months old, 258.9+/-27 g) were divided into five groups: (1) control group; (2) injured TA and analyzed 3 days later; (3) injured TA treated with ALT-C and analyzed 3 days later; (4) injured TA and analyzed 7 days later and (5) injured TA treated with ALT-C and analyzed 7 days later. The injured muscle received 25 microl of ALT-C (50 nM). The injured and uninjured muscle areas were quantified by light microscopy. The MMP activity was evaluated through zymography, and mRNA of MyoD, VEGF and MMP-2 was assessed by quantitative polymerase chain reaction. ALT-C neither reduced the muscle injury area nor altered the pattern of MyoD and VEGF expression in injured muscles. However, ALT-C reduces both MMP-2 mRNA and gelatinolytic activity in injured muscles. The study indicates that ALT-C, at the tested concentrations, did not improve muscle regeneration process in rats. The effect on MMP-2 mRNA and gelatinolytic activity suggests that ALT-C changes the overall balance of ECM protein turnover during muscle regeneration.