Motor Improvement of Skilled Forelimb Use Induced by Treatment with Growth Hormone and Rehabilitation Is Dependent on the Onset of the Treatment after Cortical Ablation

Abstract

We previously demonstrated that the administration of GH immediately after severe motor cortex injury, in rats, followed by rehabilitation, improved the functionality of the affected limb and reexpressed nestin in the contralateral motor cortex. Here, we analyze whether these GH effects depend on a time window after the injury and on the reexpression of nestin and actin. Injured animals were treated with GH (0.15 mg/kg/day) or vehicle, at days 7, 14, and 35 after cortical ablation. Rehabilitation was applied at short and long term (LTR) after the lesion and then sacrificed. Nestin and actin were analyzed by immunoblotting in the contralateral motor cortex. Giving GH at days 7 or 35 after the lesion, but not 14 days after it, led to a remarkable improvement in the functionality of the affected paw. Contralateral nestin and actin reexpression was clearly higher in GH-treated animals, probably because compensatory brain plasticity was established. GH and immediate rehabilitation are key for repairing brain injuries, with the exception of a critical time period: GH treatment starting 14 days after the lesion. Our data also indicate that there is not a clear plateau in the recovery from a brain injury in agreement with our data in human patients.

Figure 1

Schematic diagram of the experimental design. In (a), (b), and (c), −10 to 0 represent the days of training of the animals for paw-reaching-for-food task and to record the preferred forelimb (preoperative stage). In day 0, the motor cortex contraleral to the preferred limb was lesioned by aspiration. Arrows indicate the days during which GH or vehicle was given to each group of animals. Two groups of lesion animals (not GH treated) were given vehicle: in one group, it was administered on days 7 and 35 postablation (altogether called LV7/LV35 group) and in another group on day 14 (LV14 group) postablation. S indicates the days during which animals received short-term rehabilitative therapy, while L indicates the days during which animals received long-term rehabilitative therapy. 17 to 35 in (a), 21 to 35 in (b), and 17 to 35 in (c) correspond to resting period days (animals were kept in their cages without receiving any treatment). 43 correspond to the last day of long-term rehabilitative therapy.

Figure 2

Consecutive photographs illustrating a rat in the test cage showing successful responses in the paw-reaching test during training in the presurgical phase. The design of the test cage prevented use of the tongue to retrieve food pellets or to rake the pellets.

Figure 3

(a) Photograph of an example of a rat brain with motor cortex ablation (white arrow). (b) Photomicrograph of a brain coronal section showing the motor cortex lesion. Cg1: cingulated cortex, area 1; S1FL: primary somatosensory cortex, forelimb; wm: white matter. Scale bar = 100 μm.

Figure 4

(a) Immunoblot images of nestin and actin expression in the motor cortex of the undamaged hemisphere, at 51 days postablation. Results show nestin and actin expression in homogenates of motor cortex samples from animals that were under different GH treatments (LGH7, LGH14, and LGH35) or controls (CV, LV14, and LV7/35). In each immunoblot band, either nestin or actin is identified with its corresponding GH-treated animal or control, and it comes from one animal in each case. Nestin and actin molecular weight, kilodaltons (kDa), is indicated. (b) Densitometric analysis of immunoblot images of nestin and actin displayed in (a). Arbitrary units (A.U.) of densitometry. Blue points represent the values (densitometric arbitrary units from immunoblot bands) of nestin and actin expression in each motor cortex sample that comes from one animal. Red underlined ellipse identifies samples from animals treated with GH, and green underlined ellipse identifies samples from control animals.

Figure 5

Animals treated with GH or vehicle at 7 days after cortical ablation. Behavioral results obtained in the paw-reaching-for-food task with the preferred paw (impaired paw) at the presurgical phase (PRE), postablation (POST), and rehabilitative therapies. (a) Mean percentage of successful responses (successful responses/total number of responses). (b) Mean of the total number of responses (successful and unsuccessful with both paws). The rehabilitative therapies consisted in the forced use of the impaired paw, in daily sessions for 3 min during 9 consecutive days (indicated in x-axis). No differences existed between LGH7 animals and sham-operated controls (CV), while a clear lack of successful responses was found in lesion animals treated with vehicle (LV7) as compared to both GH-treated animals and controls. Significant levels are obtained after comparison with sham-operated controls (CV). β = P < 0.01 (Bonferroni's test). GH arrows indicate when GH treatment commenced and finished in LGH7 animals.

Figure 6

Animals treated with GH or vehicle at 14 days after cortical ablation. Behavioral results obtained in the paw-reaching-for-food task with the preferred paw (impaired paw) at the presurgical phase (PRE), postablation (POST), and rehabilitative therapies. (a) Mean percentage of successful responses (successful responses/total number of responses). (b) Mean of the total number of responses (successful and unsuccessful with both paws). The rehabilitative therapies consisted in the forced use of the impaired paw, in daily sessions for 3 min during 13 (short-term rehabilitation) and 9 (long-term rehabilitation) consecutive days (showed in x-axis). Significant levels (the Bonferroni post hoc test) indicated that both vehicle-treated animals (LV14) and GH-treated rats did not improve their percentage of successful responses in comparison to results obtained in the sham-operated control group (CV). β = P < 0.01; GH arrows indicate when GH treatment commenced and finished in LGH14 animals.

Figure 7

Animals treated with GH or vehicle at 35 days after cortical ablation. Behavioral results obtained in the paw-reaching-for-food task with the preferred paw (impaired paw) at the presurgical phase (PRE), postablation (POST), and rehabilitative therapies. (a) Mean percentage of successful responses (successful responses/total number of responses). (b) Mean of the total number of responses (successful and unsuccessful with both paws). The rehabilitative therapies consisted in the forced use of the impaired paw, in daily sessions for 3 min during 9 consecutive days indicated in (x-axis). GH-treated animals (LGH35) improved their percentage of successful responses after the second session, reaching a value no longer different to that of the sham-operated control group (CV), while vehicle-treated animals (LV35) did not change their low percentage of successful results. Significant levels are obtained after comparison with sham-operated controls (CV). β = P < 0.01; Δ = P < 0.05 (Bonferroni's test). GH arrows indicate when GH treatment commenced and finished in LGH35 animals.