Combining glial cell line-derived neurotrophic factor gene delivery (AdGDNF) with L-arginine decreases contusion size but not behavioral deficits after traumatic brain injury

Abstract

Our laboratory has previously demonstrated that viral administration of glial cell line-derived neurotrophic factor (AdGDNF), one week prior to a controlled cortical impact (CCI) over the forelimb sensorimotor cortex of the rat (FL-SMC) is neuroprotective, but does not significantly enhance recovery of sensorimotor function. One possible explanation for this discrepancy is that although protected, neurons may not have been functional due to enduring metabolic deficiencies. Additionally, metabolic events following TBI may interfere with expression of therapeutic proteins administered to the injured brain via gene therapy. The current study focused on enhancing the metabolic function of the brain by increasing cerebral blood flow (CBF) with l-arginine in conjunction with administration of AdGDNF immediately following CCI. An adenoviral vector harboring human GDNF was injected unilaterally into FL-SMC of the rat immediately following a unilateral CCI over the FL-SMC. Within 30min of the CCI and AdGDNF injections, some animals were injected with l-arginine (i.v.). Tests of forelimb function and asymmetry were administered for 4weeks post-injury. Animals were sacrificed and contusion size and GDNF protein expression measured. This study demonstrated that rats treated with AdGDNF and l-arginine post-CCI had a significantly smaller contusion than injured rats who did not receive any treatment, or injured rats treated with either AdGDNF or l-arginine alone. Nevertheless, no amelioration of behavioral deficits was seen. These findings suggest that AdGDNF alone following a CCI was not therapeutic and although combining it with l-arginine decreased contusion size, it did not enhance behavioral recovery.

Figure 1

Combining AdGDNF and L-arginine Significantly Reduces Contusion Size. A) Coronal section of an animal that received a CCI, but received no treatment post injury. B) Animal received a CCI and was treated with AdGDNF and L-arginine immediately post-injury resulting in a significantly smaller contusion. CCI=Contusion cavity; CC = Corpus Callosum; ST=Striatum; LV= Lateral Ventricle; Scale bar (A&B) = 250μm C) Volume of Remaining Cortex - Animals treated with AdGDNF and L-arginine post-CCI had significantly smaller contusions (greater amount of remaining cortex) than animals in the CCI group, which did not receive any treatment; as well as injured animals treated with AdGDNF or L-arginine alone (*p<0.05 significantly different from sham; †significantly different from CCI, CCI+L-arg, CCI+AdGDNF p<0.05)

Figure 2

Foot Fault Test. All CCI groups had significantly larger deficits in motor coordination than the Sham group (*p<0.05), yet there were no significant differences between the CCI groups. These deficits began to recover over time but did not reach uninjured Sham levels by day 28.

Figure 3

Total Ipsilateral Forelimb Preference. Injured animals treated with AdGDNF, L-arginine, or both preferred the ipsilateral forelimb when compared to Shams (*p<0.05). There were no significant differences between the CCI groups.

Figure 4

Wall Ipsilateral Forelimb Preference. Injured animals that received no treatment, treated with AdGDNF, L-arginine, or both preferred the ipsilateral forelimb when compared to Shams (*p<0.05). There were no significant differences between CCI groups.

Figure 5

GDNF expression –GDNF protein expression is robust in Sham animals one week post-injection, producing a penumbra of staining (outlined area in A) and significant numbers of GDNF+ Cells (B). Following injury, GDNF expression decreases significantly, with slightly more expression seen in the animals treated with AdGDNF+L-Arginine (C) versus AdGDNF alone (D). Following injury, GFP+ Cells are still present surrounding the contusion cavity (E). Scale bar - A,C, D, &E = 250μm; B=25 μm. Arrows point to GDNF penumbra and or GDNF+cells. LV=Lateral Ventricle; CC = Corpus Callosum; ST=Striatum; CTX =Cortex; CCI=Contusion cavity