Mechanical and microstructural changes of skeletal muscle following immobilization and/or stroke

Abstract

Patient management following a stroke currently represents a medical challenge. The presented study investigates the effect of immobilization on skeletal muscles in short positions after a stroke. A rat model was implemented in order to compare four situations within 14 days including control group, immobilization of one forelimb without stroke, stroke without immobilization and stroke with immobilization of the paretic forelimb. To analyze the changes of the mechanical properties of the passive skeletal muscle, the biological tissue is assumed to behave as a visco-hyperelastic and incompressible material characterized by the first-order Ogden’s strain energy function coupled with second-order Maxwell’s model. The material parameters were identified from inverse finite element method by using uniaxial relaxation tests data of skeletal muscle samples. Based on measurements of histological parameters, we observe that muscle immobilization led to microconstituents changes of skeletal muscles that were correlated with degradations of its mechanical properties. In the case of immobilization without stroke, the neurological behavior was also altered in the same manner as in the case of a stroke. We showed that immobilization of skeletal muscles in short positions produced contractile tissue atrophy, connective tissue thickening and alteration of passive mechanical behavior that were more damaging than the effects produced by a stroke. These results showed then that immobilization of skeletal muscles in short positions is highly deleterious with or without a stroke.

Appendix

See Fig. 7.

Fig. 7

a Diagram, b schema and c photos illustrating the methods for carrying out MCAo and immobilization. ACA, anterior cerebral artery; MCA, middle cerebral artery; Pt, pterygo-palatine artery; ECA, external carotid artery; ICA, internal carotid artery; CCA, common carotid artery

The model was done under isoflurane anesthesia (5% for induction, 2.25–2.5% for maintenance via artificial ventilation in 30% oxygen and 70% nitrous oxide at l L/min). Surgical procedures were carried out as previously described (Ouk et al. 2009). First, having exposed the right carotid, it consists of ligating pterygopalatine, common carotid and external carotid arteries with silk sutures. Then, a temporary knot and an aneurismal clip were placed successively downstream of the puncture site on the common carotid artery. A 4/0 monofilament nylon was introduced through the right common carotid artery. Once filament got to the aneurysm clip, the knot was tightened and the clip was removed. Finally, the monofilament was gently advanced of almost 20–22 mm into the internal carotid artery until resistance is felt, meaning that the rounded head of monofilament reached the middle cerebral artery. The monofilament rounded head was obtained by burning the filament end and optimized for a diameter of. After 60 min, the monofilament was removed until the rounded head was blocked by the ligature. All the silk sutures and the monofilament were left in place. Note that body temperature was maintained at by a heated surgery table. The recovering place is a controlled room at 27° with an oxygen adjunction. Then, once awake, rats were placed in a cage at ambient temperature with the possibility to eat and drink ad libitum. It should be underlined that the control operation (for control and immobilized groups) consists in 60-min anesthesia and same handling without advance the monofilament until the obstruction.

See Figs. 8, 9, 10 and 11.

Fig. 8

Illustration of the semiautomatic collagen analysis. a Initial part of red Sirius slice. b Analyzed slice from k-mean clustering (k = 4) with endomysium in blue and perimysium in red. c Cluster including muscle fiber. d Cluster including white background. e Cluster including endomysium f Cluster including perimysium. In order to illustrate the process, the endomysium and perimysium cluster was colored in blue and red, respectively. Some misallocations remain after first step (k-mean clustering). That is to say endomysium in perimysium cluster and vice versa. There were manually corrected by the user with a polygonal tool

Fig. 9

Illustration of the semiautomatic immunohistology analysis. a Cross section incubated in primary antibody collagen against MHCI and MHCII. b Slice subdivision with selected region of interest (ROI) of one myofiber. Each myofiber was selected and a mean {Green;Blue} doublet was computed inside each ROI. c Myofiber automatically assigning using a K-mean clustering (k = 3)

Fig. 10

Diagram of the inverse finite element method

Fig. 11

a Correlation between biceps brachii wet weight and body weight. b Correlation between muscle wet weight and cerebral infarct volume. Pearson coefficient (r, p) was indicated for all groups (black), for immobilized group (orange) or non-immobilized group (blue)

Whether it is for all groups (black solid line), for immobilized group (orange dotted line) or non-immobilized group (blue dotted line), we observe a strong correlation between biceps brachii wet weight and body weight (Fig. 8a). Furthermore, we show that muscle weight and cerebral infarct size are negatively correlated as previously described by Parkkinen et al. (2013) (Fig. 8b).