A novel approach to induction and rehabilitation of deficits in forelimb function in a rat model of ischemic stroke

Abstract

Aim: Constraint-induced movement therapy (CIMT), which forces use of the impaired arm following unilateral stroke, promotes functional recovery in the clinic but animal models of CIMT have yielded mixed results. The aim of this study is to develop a refined endothelin-1 (ET-1) model of focal ischemic injury in rats that resulted in reproducible, well-defined lesions and reliable upper extremity impairments, and to determine if an appetitively motivated form of rehabilitation (voluntary forced use movement therapy; FUMT) would accelerate post-ischemic motor recovery.

Methods: Male Sprague Dawley rats (3 months old) were given multiple intracerebral microinjections of ET-1 into the sensorimotor cortex and dorsolateral striatum. Sham-operated rats received the same surgical procedure up to but not including the drill holes on the skull. Functional deficits were assessed using two tests of forelimb placing, a forelimb postural reflex test, a forelimb asymmetry test, and a horizontal ladder test. In a separate experiment ET-1 stroke rats were subjected to daily rehabilitation with FUMT or with a control therapy beginning on post-surgery d 5. Performance and post-mortem analysis of lesion volume and regional BDNF expression were measured.

Results: Following microinjections of ET-1 animals exhibited significant deficits in contralateral forelimb function on a variety of tests compared with the sham group. These deficits persisted for up to 20 d with no mortality and were associated with consistent lesion volumes. FUMT therapy resulted in a modest but significantly accelerated recovery in the forelimb function as compared with the control therapy, but did not affect lesion size or BDNF expression in the ipsilesional hemisphere.

Conclusion: We conclude that refined ET-1 microinjection protocols and forcing use of the impaired forelimb in an appetitively motivated paradigm may prove useful in developing strategies to study post-ischemic rehabilitation and neuroplasticity.

Figure 1

Overview of the surgical procedure. (A) Approximate targeted infarct regions based on microinjection locations (in black; adapted from the Rat Brain Atlas). (B) Gross assessment of the lesion. (C) Typical damage produced at targeted stereotaxic levels. (D) Volume of the total infarct, as well as the cortical and striatal contribution. (E) Infarct distribution across stereotaxic levels. Error bars represent SEM. n=14 (ischemia), n=6 (sham).

Figure 2

Effects of the ET-1 microinjection protocol on various tests of contralesional forelimb function in Experiment 1. (A) Tactile-stimulated forelimb placing test of reflexive paw placement following physical stimulation. (B) Vibrissae-stimulated forelimb placing test of reflexive paw placement following physical-sensory stimulation. (C) Forelimb postural reflex test of reaching for a flat surface during descent. (D) Cylinder test of forelimb use for upright support. Error bars represent SEM. n=14 (stroke), n=6 (sham); ^bP<0.05 relative to sham.

Figure 3

Rehabilitation intensity. Mean (±SEM) time (min) spent moving the exercise ball each day during 30 min of rehabilitation. On PSD 5, Sham animals spent 18±1.5 min (SEM) engaging in the voluntary forced movement, while ET-1 animals spent 19±1.5 min (SEM) moving. At PSD 21, both groups spent 12±1.7 min moving. n=11 (stroke); n=12 (sham).

Figure 4

Effects of the ET-1 microinjection protocol on various tests of contralesional forelimb function in Experiment 2. (A) Tactile-stimulated forelimb placing test. (B) Forelimb postural reflex test. (C) Cylinder test. (D) Horizontal ladder test. Error bars represent SEM. n=11 (stroke groups), n=24 (combined sham group); ^bP<0.05 relative to sham.

Figure 5

BDNF expression. (A) Representative images of BDNF expression in the ipsi- and contralesional hemisphere. (B) Quantification of the percent cells expressing BDNF in stroke and sham treated rats. Following surgery, Sham animals showed BDNF expression from 31.6%±2.7% (SEM) cells, compared to Stroke animals with 40.1%±3.4% expressing the protein. In the contralesional hemisphere there were no significant differences in percent BDNF expression, although there was a tendency for stroke animals to have greater percent expression [Sham=32.2%±4.8% (SEM); Stroke=41.0%±2.2%; P>0.05). n=4; ^bP<0.05.