Histological and functional assessment of the efficacy of constraint-induced movement therapy in rats following neonatal hypoxic-ischemic brain injury

Abstract

Constraint-induced movement therapy (CIMT) is used in stroke rehabilitation to promote recovery of upper limb motor function. However, its efficacy in improving functional outcomes in children with hemiplegic cerebral palsy has not been clearly determined in clinical or experimental research. The aim of our study was to assess the efficacy of a new experimental model of CIMT, evaluated in terms of mortality, stress, motor and cognitive function in rats having undergone a neonatal hypoxic-ischemic (HI) brain injury. Neonatal HI injury was induced at post-natal day 7 through unilateral ligation of the common carotid artery followed by exposure to hypoxia for 2 h. CIMT was implemented at 3 weeks, post-HI injury, using a pouch to constrain the unimpaired forelimb and forcing use of the affected forelimb using a motorized treadmill. After HI injury, animals demonstrated motor and cognitive deficits, as well as volumetric decreases in the ipsilateral hemisphere to arterial occlusion. CIMT yielded a modest recovery of motor and cognitive function, with no effect in reducing the size of the HI lesion or post-HI volumetric decreases in brain tissue. Therefore, although animal models of stroke have identified benefits of CIMT, CIMT was not sufficient to enhance brain tissue development and functional outcomes in an animal model of hemiplegic cerebral palsy. Based on our outcomes, we suggest that CIMT can be used as an adjunct treatment to further enhance the efficacy of a program of rehabilitation in children with hemiplegic cerebral palsy.

Keywords: behavioral recovery; constraint-induced movement therapy; hypoxic-ischemic brain injury; neonatal rat; rehabilitation.

Figure 1.

Effect of CIMT on body weight and plasma corticosterone levels. (A and B) A CIMT-treated rat, with restraint to the unaffected forelimb by wearing a pouch, forcing the use of the affected limb on a motorized treadmill. (C) Timeline of the experimental design. (D) Body weight was monitored from 3 weeks after HI injury, and measured up to 6 weeks after HI injury. Groups were as follows: HI, HI injury and non-treated group (HI/vehicle: n=12); 10, HI injury and CIMT for 10 min (n=12); 20, HI injury and CIMT for 20 min (n=12); 30, HI injury CIMT for 30 min (n=12). (E) Corticosterone concentration in plasma was determined by ELISA assay at 7 weeks after HI. *P<0.05 vs. sham group (unpaired t-test). HI, hypoxic-ischemic; CIMT, constraint-induced movement therapy.

Figure 2.

Effect of CIMT on cylinder test and rota-rod test. (A) The use of forelimb for support during weight bearing behavior was assessed through examination of preference for the ipsilateral forelimb use by the cylinder test. (B) Motor coordination and balance was assessed by the rota-rod test. *P<0.05 vs. sham group (unpaired t-test). HI, hypoxia-ischemia; CIMT, constraint-induced movement therapy.

Figure 3.

Effect of CIMT on open-field test. Locomotor and exploratory behaviors were evaluated using the open-field test. After 5 min of adaptation in a black box, (A) total distance traveled and (B) resting time were video-recorded for 15 min. HI, hypoxia-ischemia; CIMT, constraint-induced movement therapy.

Figure 4.

Effect of CIMT on passive avoidance task. The cognitive function and memory acquisition were assessed by passive avoidance task at 7 weeks after HI. (A) In the training trial, the rats were placed in the illuminated compartment, and the step-through latency for animals to enter the dark compartment was measured. (B) The retention trial was performed 24 h after the acquisition trial in the same manner and the time taken to re-enter the dark compartment was recorded. *P<0.05 vs. sham group (unpaired t-test). HI, hypoxia-ischemia; CIMT, constraint-induced movement therapy.

Figure 5.

Effect of CIMT on volume atrophy. (A) Gross assessment of the HI injury lesion. (B) Photomicrograph of cresyl violet staining. Each region was located at 1.28, −3.48 and −5.28 mm from the bregma. Scale bar, 4 mm. (C and D) Histogram of the cortex, striatum, hippocampus and midbrain for histological analysis. *P<0.05 and **P<0.01, vs. sham group (unpaired t-test). HI, hypoxia-ischemia; CIMT, constraint-induced movement therapy.

Figure 6.

Effect of CIMT on proliferation in the subgranular zone and subventricular zone. BrdU staining in the (A-C) SGZ and (D-F) SVZ at 7 weeks after HI. Quantification graphs of BrdU-positive cells (white arrows) in (C) SGZ and (F) SVZ. *P<0.05 vs. sham group (unpaired t-test). The red rectangle illustrates the imaging field. DAPI (blue) for nuclei. Scale bar, 20 µm. CIMT, constraint-induced movement therapy; SGZ, subgranular zone; SVZ, subventricular zone; HI, hypoxia-ischemia.