The neuroprotective effects of adalimumab on rats with experimental peripheral nerve injury: an electron microscopic and biochemical study

AbstractAdalimumab, a new generation anti-inflammatory agent, exerts its effect through tumor necrosis factor alpha (TNF-α) which is secreted from immune response cells like macrophages or lymphocytes. TNF-α was proved to have an important role in apoptosis and demyelinization process and blockage of its activity may improve neural healing. Here we investigate probable neuroprotective influence of adalimumab in a rat on peripheric nerve injury model with biochemical and electron microscopic methods.

Methods:Forty adult Wistar albino rats were divided into sham, sciatic nerve trauma, low dose adalimumab and high dose adalimumab groups at random. Six rats from each group were reserved from biochemical and remaining 4 rats for electron microscopy. Neural injury was induced with clip compression after dissection of sciatic nerves and adalimumab was injected at the same time. Rats were sacrificed after 2 weeks of adalimumab treatment period.

Results:Nerve tissue lipid peroxidation values were found to be statistically significant decreased both in low dose and high dose adalimumab treatment groups when compared with rats subjected to sciatic nerve trauma only.

ConclusIon:The results of this study showed that adalimumab is an effective neuroprotective agent for neural healing particularly in the early phase.

INTRODUCTION Peripheral nerve injuries are common andimportant causes of long term morbidity.[1] The incidence is approximately2.8% among all cases of trauma.[2]Upon exposure to nerve compression, there may be a spectrum of funcitonal losses from a slight weakness to total paralysis of muscles or from total loss of sensation to a mild paraesthesias. Degree of severity is related to the involved nerve and location, pressure level and duration of the compression. Even after relieving the compression, nerve damage may still continue.[3]Because of ischemic reperfusion injury,responsible from the major damage to the nervous tissue.[4]Ischemic reperfusion injury is mainly initiated by free oxygen radicals and inflamatory reactions, caused by reduction reactions and neurtrophilic infiltrations, respectively.[5]Contemporary management of peripheral nerve injuriesencompasses non-steroidal anti-inflammatory drugs, steroids, nerve growth factors, thyroid hormones, growth hormone, ACTH, and insulin-like peptides.[6,7]In this study, effects of a monoclonal antibody agent, adalimumab, on injured nerve tissue was investigated on an experimental clip compression injury model in arat sciatic nerve.

MATERIALS AND METHODS40 male Wistar Albino rats, of body weights ranging from 180-210 gr, ages between 3 to 5 months were used for the study. The rats were housed in cages (accomodating 4 rats) in an air conditioned room with controlled temperature (18-21 degrees Celcius) and automatic lightening (alternating 12-hour periods of light and dark). Food and water were given ad libitum. Rats were randomly categorized into 4 groups: sham group, trauma group, low-dose and high dose of adalimumab groups. The rats were kept overnight fasting and weighed before performing surgical proceduresunder general anesthesia. Rats were anesthesized intraperitoneally by mixture of 10mg\kg Xylocaine (Rhompun ®, a 2% solution, Bayer, Istanbul) and 50 mg\kg ketamine hydrochloride (Ketalar ®, a 5% solution, Parke Davis Pharmaceutical Industries under the license Eczacıbaşı, Levent, Istanbul, Turkey). Anesthesized rats were placed prone position with the extremities attached aside. Superficial sterilization was provided by polyvinylpyrolidone iodine (Polyod ®, a 10% solution, Drogsan Pharmaceutical Industry, Delhi) covering sacral area and both lower extremities. In right lower extremity, a longitudinal skin incision from trochanter level was performed. Following dissection, sciatic nerve was exposed by incising the gluteus maximus muscle vertically. Sciatic nerve were meticulously dissected by preserving perineurium, without any tractional injury. The nerve was compressed for 2 minutes with a temporary aneurysm clip, closing force of 50g\cm2 (Yasargil FE 693 temporary aneurysm Clip- Aesculap). The injured region was marked proximally and distally with polypropylene thread (Prolene). These procedures were repeated for the rats in each group.The Rats in low dose and high dose groups were given adalimumab three times intraperitoneally, during surgery and at 1 and 2 weeks of the injury, in 5mg\kg and 50 mg\kg doses, respectively.Two weeks later, the rats are sacrificed by high dose of anesthesia and sciatic nerve segments were removed. Tissue samples were cut into 1mm3 pieces and kept in0.1M phosphate buffered 2,5% gluteraldehite (pH 7,4) solution for 2 h fixation. After washing 3 times with a buffered solution, the samples were exposed to 1% osmium tetroxide for 1 hour for postfixation. Later, they are dehydrated in alcohol.Lastly, the samples processed with propylene oxide and blocs were prepared with embedding material by using Araldite CY212 kit. These blocks were polymerized in 560 degree Celsius oven for 48 hours, then semi-thin sections were optained and stained with toluidine blue for examining under light microscopy. From the assigned region, thin sections were taken and stained with uranyl acetate - lead citrate in order to examine under a microscope with EVO LS10 transmission attachment.In this study, measurement of thiobarbituric acid reactive components were used to ascertain lipid peroxidation as defined by Uchiyama and Mihara.[8]Tissue samples were homogenized in potassium phosphate buffer solution 1:10 (w:v) by using Dounce homogenization. Total of 0,5 ml homogenate was first stirred with 3 mL of 1% phosphoric acid, then 1 ml of 0.67% of TBA was added. Later, the tubes were suspended in boiling water for 45 minutes. After cooling, thiobarbituric acid components were seperated into butanole and absorption values were determined at 532 nm. Taking these values into consideration, thiobarbituric acid reactive components-MDA complex base molar absorption value was determined as 0.156x105 M-1 cm-1and level of lipid peroxide was calculated as nanomoles per gram of tissue.