Neurology India Medknow Publications on behalf of the Neurological Society of India ISSN: 0028-3886 EISSN: 1998-4022 Vol. 58, Num. 2, 2010, pp. 195-200

Neurology India, Vol. 58, No. 2, March-April, 2010, pp. 195-200

Original Article

db-Cyclic adenosine monophosphate promotes axon regeneration and motor function recovery in cerebral ischemia-reperfusion rats

Lingchuan Niu, Jia Zhou, Ying Huang, Yan Chen, Changqing Li

Department of Neurology, The Second Affiliated Hospital of Chongqing University of Medical Sciences, Chongqing 400010, China

Correspondence Address: Changqing Li, Department of Neurology, 2nd Hospital, Chongqing University of Medical Sciences, Chongqing-400 010, China, lichangqing-li@163.com

Date of Acceptance: 23-Oct-2009

Code Number: ni10055

PMID: 20508335

DOI: 10.4103/0028-3886.63786

Abstract

Background: There is no effective axon regeneration in adult mammalians.
Objective: To investigate the effects of dual-acid cyclic adenosine monophosphate (db-cAMP) on the axon regeneration, motor function recovery and RhoA signal pathway in cerebral ischemia-reperfusion rats, and to explore the possible clinical application and mechanism.
Materials and Methods: Middle cerebral artery ischemia-reperfusion model was established by nylon monofilament occlusion method in 105 Sprague-Dawley (SD) rats. Semi-quantitative Western blot analysis was used to assess protein expression level of growth-associated protein-43 (GAP-43) and RhoA. Montoya staircase test score was used to test the motor function of affected forelimb.
Results: Compared to the ischemia group, the staircase test score in the db-cAMP group was increased significantly at 30-day (P < 0.05), and GAP-43 protein expression in the db-cAMP group was enhanced significantly at 7-day and 14-day (P < 0.05), and RhoA protein expression in the db-cAMP group was decreased significantly between 24 h to 14-day (P < 0.01).
Conclusion: These results show that db-cAMP can promote axon regeneration and the recovery of motor function by inhibiting RhoA signal pathway.

Keywords: Axon regeneration, cerebral ischemia, dual-acid cyclic adenosine monophosphate, growth associated Protein-43, RhoA.

Introduction

Axonal regeneration is difficult when injury of adult mammalian central nervous system (CNS) occurs, thus limiting the functional recovery. However, several animal experiments, both in vivo and in vitro, have shown that if the central branch of the dorsal root ganglion (DRG) neurons is damaged followed by injuries in the peripheral branch one week later, extensive axon regeneration into and beyond the lesion site can be observed, the phenomenon known as "conditioning lesion" effect. ^[1],[2] Qiu and colleagues ^[3] have shown that one day post-peripheral-lesion, the level of cyclic adenosine monophosphate (cAMP) in the DRG neurons increased threefold. An intriguing possibility is that this "conditioning lesion" results in an elevation of cAMP, that allows for the improved central-branch regeneration. Use of the spinal cord injury model (central branch of DRG) has suggested the possible mechanism for the promotive effect of cAMP on the axon regeneration. ^[4],[5],[6] Protein kinase A (PKA) is activated by cAMP in cells and PKA antagonizes the inhibitory effect of RhoA on the axon regeneration. RhoA signal pathway is also the working pathway of nerve growth inhibitors including Nogo-A, ^[7], myelin-associated glycoprotein (MAG) ^[8] and oligodendrocyte-myelin glycoprotein (OMgp). ^[9] All of them have the same receptor complex (NgR- P75NTR). Rho A kinase (ROCK) is the downstream effector of RhoA which regulates the activities of actin cytoskeleton and causes the collapse of axonal growth cone thereby inhibiting axon regeneration. ^{[10],[11],[12]}

cAMP cannot penetrate the membrane of the cell easily and it is hydrolyzed by the phosphodiesterase in cells quickly. db-cAMP is a membrane-permeable derivative of cAMP and it can confront the destructive effect of phosphodiesterase. The response speed of db-cAMP is more rapid than cAMP and the response time of db-cAMP lasts longer than cAMP. It has the same physiological effect in vivo as cAMP. The purpose of this study is to observe whether exogenous db-cAMP can promote axon regeneration and motor function recovery in adult rats with cerebral ischemia-reperfusion injury, and how does db-cAMP induce axon regeneration? Is it associated with RhoA signal pathway? Growth associated protein (GAP-43) is elected as one of the molecular probes for the study of axon regeneration because it has high expression in neurons during development and regeneration.

Materials and Methods

Experimental plan

One hundred and five healthy male adult Sprague-Dawley (SD) rats, weighing 200-250 g, were provided by the Experimental Animal Center of Chongqing Medical University [SCXK (Yu) 2007-0001]. The experiment was approved by the Laboratory Animal Management Committee of Chongqing Medical University, and the whole procedure was in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals by the Ministry of Science and Technology of China. ^[13] The rats were randomly divided into five subgroups: normal group (n=5), sham-operated group (n=25), cerebral ischemia-reperfusion group (ischemia group, n=25), normal saline (NS) group (n=25), db-cAMP group (n=25). Four rats from each group were sacrificed at different sampling times post surgery: 6h 24h, 48h 7-day and 14-day. Five rats were tested for forelimb function at 30-day post-surgery in each group.

Animal model for cerebral ischemia-reperfusion

The rats in ischemia, NS and db-cAMP groups were made into models of middle cerebral artery ischemia-reperfusion according to the method described by Longa: ^[14] First, a middle cervical incision was made, then the right common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA) were separated before the distal end of the ECA was ligated. The stump of ECA was pulled down to get a straight line with ICA and the ICA and CCA were clipped with microvascular folder temporarily. A small opening was made on the ECA stump near the bifurcation, the line-emboli (fishline 0.23 mm in diameter, coated with 4 mm of paraffin wax on the top) was inserted into the ICA through the ECA, then the microvascular folder on ICA was released so as the emoboli could be continued to insert about 18-20 mm for ischemia of the middle cerebral artery (MCA). The emboli were then fixed in the ICA with silk thread; the microvascular folder of CCA was loosened and the incision was sutured. The embolus was drawn out 2 h later for reperfusion. The criteria for a successful model were the paralysis of the left limb when the rat has gained consciousness post-surgery. In sham-operated group, the embolus was inserted to a distance of 12 mm and was pulled out immediately. The rats in the normal group received no treatment.

Ventricle perfusion

The successfully modeled rats in NS and db-cAMP groups were anesthetized intraperitoneally with chloral hydrate (35 mg/100 g), sagittal incision was made at the top of the crown, then the needle was applied 0.8 mm posterior and 1.5mm lateral of the former fontanelle in the right skull at 3.5 mm depth. NS group: 10 μl sterile NS was injected into ventricle slowly; db-cAMP group: 0.5 g db-cAMP/NS 10 μl was injected into the ventricle slowly (15 min). The syringe then was removed, the eye of the needle was blocked by bone wax and the scalp incision was then closed by suturing.

Main outcome measures

Forelimb function tests in rats

The rats were tested for forelimb function using Montoya staircase test score. ^[15] They were trained for staircase test seven days before surgery to determine the preference of rat claw (which of the forelimbs was preferred to crawl for food). One hundred and five rats with left-claw preference were used for the subsequent experiments. Rats were tested at 1-day before surgery and again when rats were sober, and at 14-day and 30-day post-surgery. Rats must fast for 12 h and drink freely before every test. Montoya staircase test score: Only the left side of the stairs stored food which was added once every 5 min, the maximum amount of food was 72 balls (45 mg/1 food ball). The number of balls eaten by rats in 1-h observation was the score. One person who was unaware of the respective treatment groups observed each rat for 1 h.

Western-blot

The precise procedure of semi-quantitative Western-Blot analysis is as described previously. ^[16] Rats were killed at the indicated time and 0.1 g of the right side of cerebral cortex was homogenized. Protein extract followed the manufacturer′s protocol. The whole protein extract was preserved at -70°C and to avoid repeated freeze-thaw. The protein contents were determined by Bradford protein assay. Proteins were separated by 12% sodium dodecyl sulfate polyacrylamidegel electropheresis (SDS-PAGE), GAP-43 and RhoA protein expressions were analyzed using GAP-43 specific antibody (1:1000, Chmicon, USA) and RhoA-specific antibody(1:500, Santa,USA). GAPDH (38KD, Zhongshan Golden Bridge Inc., Beijing, China) was used as reference. After electrophoresis of the samples, proteins were electroblotted onto polyvinylidene fluoride (PVDF) membranes. The blots were washed with TBS, membranes were incubated with the GAP-43 (43KD) / RhoA (24KD) antibody for 16-18 h at 4 (?). Then the blots were washed with TBST and TBS, followed by incubation with the secondary antibody for 1 h at 37 (?) After washing, proteins were visualized by electrochemiluminescence (ECL) method. The blot membranes was scanned with an imaging scanner (chemiDoc XRS) and analyzed with the Quantity One 4.6 imaging software (Bio-rad).

Statistical analysis

Data were analyzed using SPSS 11.0 software. Data were expressed as mean ± standard deviation (SD). Statistical analysis was performed by ANOVA to compare two or more groups. P < 0.05 was regarded as statistically significant.

Results

Forelimb functional tests score

The staircase test scores of rats in the five groups were similar before surgery (P > 0.05). There was no activity of the affected limb when rats were sober post-surgery and the motor function slowly recovered over time. In the db-cAMP group, the score was higher than the ischemia and NS group at 14-day and 30-day and it was statistically significant at 30-day (P < 0.05). This showed that giving exogenous db-cAMP (ventricle perfusion) improved motor function recovery in rats with cerebral ischemia [Figure - 1],[Table - 1].

GAP-43 protein expression

Compared to the normal and sham-operated groups, GAP-43 protein expression in the ischemia group increased at 24 h post-surgery then peaked at 7-day and gradually decreased to a level which was still higher than sham-operated group at 14-day. This showed that cerebral ischemia-induced GAP-43 protein expression increased in ischemic brain tissue and it continued till convalescence. GAP-43 expression in the NS and db-cAMP groups at different time points post-surgery showed the same changing trend, but its expression in db-cAMP group was higher than that in the ischemia and NS groups at each time points. It was statistically significant at 7-day and 14-day (P < 0.01/0.05), which showed that giving exogenous db-cAMP obviously enhanced GAP-43 protein expression in brain tissue after cerebral ischemia [Figure - 2] and [Figure - 3],[Table - 2].

RhoA protein expression

Compared to the normal and sham-operated groups, RhoA protein expression in the ischemia group increased at 24 h after surgery, peaked at 48 h, and gradually decreased to a level which was still higher than the sham-operated at 14-day. This showed that cerebral ischemia-induced RhoA protein expression increased in ischemic brain tissue and it continued till convalescence. RhoA expression in the ischemia and db-cAMP group at different time points post surgery showed the same changing trend, but its expression in the db-cAMP group was significantly lower than the ischemia group between the time of 24 h to 14d (P< 0.01), which showed that giving exogenous db-cMAP obviously decreased RhoA expression in brain tissue after cerebral ischemia [Figure - 4],[Figure - 5],[Table - 3].

Discussion

cAMP is an important second messenger in signaling pathways that regulate cellular processes involved in development and regeneration. Its physiological effect is cAMP-dependent protein kinase A (PKA)-dependent. Recent studies ^[4],[5],[6] have shown that cAMP can affect axonal growth via PKA in many aspects, such as regulation of inhibitors of axonal growth, accuracy of axonal growth cone sprouting and axon projection into the target area. A high level of cAMP inside the cells promotes the progress and turning of axonal growth cone and guides axons to continue to grow. Inhibiting activity of PKA would inhibit sprouting of axonal growth cone in vivo, Thus, using cAMP to regulate the activity of PKA may be a treatment target for axon regeneration and functional rehabilitation following CNS injury. In this study, db-cAMP was injected into the ventricle of adult rats with middle cerebral artery ischemia-reperfusion. The score dramatically increased in db-cAMP group at 30-day when compared with the ischemia and NS groups. This showed that db-cAMP can improve functional recovery suffered from CNS injury. These results provided a new direction in treating axon damage of CNS.

GAP-43 is a specific membrane phosphoric acid protein located in neuronal growth cone, which has been implicated in growth cone formation. It influences growth cone attachment, extension and resisting effect of retraction, signal transduction, promoting neurotransmitter release and maintaining the long-term potentiation effect. It plays a key role in guiding axon growth and regulating formation of new contacts for the axons. ^[17],[18] GAP-43 protein expression in the areas around the ischemia (ischemic penumbra) increased at 24 h, peaked at 7-day, and continued for at least 14-day in the ischemia group. This showed that neurons of the ischemic penumbra were trying to grow new axons to rebuild damaged nerve function. These results were in accordance with other reports. ^[19],[20] GAP-43 protein expression in the db-cAMP group was higher than in the ischemia group, which showed that giving exogenous db-cAMP (ventricle perfusion) promotes axon regeneration, one of the mechanisms for promoting motor functional rehabilitation.

The plasticity of the survived brain neurons has been enhanced to compensate the loss of the neural function of infarction tissue. This forms the basis for neuron-rehabilitation after a stroke. Current researches have shown that RhoA signal pathway is the same working pathway of nerve growth inhibitors including MAG, Nogo and Omgp. ^{[21],[22],[23]} RhoA kinase (ROCK) is the downstream effector of RhoA which regulates the activities of actin cytoskeleton and causes collapse of axonal growth cone and thereby inhibits axon regeneration. ^[11],[24] In this study, RhoA protein expression in the db-cAMP group was significantly lower than the ischemia and NS group between the 24 h to 14-day period. This showed that giving exogenous db-cAMP (ventricle perfusion) inhibits the activity of the RhoA signal pathway and suppresses GAP-43 protein expression. These results showed that db-cAMP can promote axon regeneration and motor function recovery through regulating the activity of the RhoA signal pathway.

Acknowledgments

This work was supported by the Natural Science Foundation of Chongqing city (Grant No. 2004-54-83).

References

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