Temporal and spatial distribution of the aquaporin 1 in spinal cord and dorsal root ganglia after traumatic injuries of the sciatic nerve

Yasemin Kaya 0 2 Umut Ozsoy 0 2 Necdet Demir 0 2 Arzu Hizay 0 2 L. Bikem Suzen 0 2 Doychin N. Angelov 0 2 Levent Sarikcioglu 0 2 0 N. Demir Department of Histology and Embryology, Akdeniz University Faculty of Medicine , Antalya 07070, Turkey 1 ) Department of Anatomy, Akdeniz University Faculty of Medicine , Antalya 07070, Turkey 2 D. N. Angelov Anatomical Institute, University of Cologne , Cologne, Germany Purpose The aquaporin family comprises a large family of integral membrane proteins that enable the movement of water and other small, neutral solutes across plasma membranes. Although function and mechanism of aquaporins in central nervous system injury have been reported, the pathophysiologic role of aquaporin 1 (AQP1) in peripheral nerve has not been extensively documented. In the present study, we aimed to study the temporal and spatial distribution of AQP1 in spinal cord and dorsal root ganglia after sciatic nerve injury. Methods Forty-eight adult female mice were randomly divided into four groups (intact controls, sham operated, cut injury, and crush injury). Animals receiving cut or crush injuries were sacrificed at the 2nd, 24th, and 48th postoperative hours. Spinal cord samples at the level of lumbosacral intumescences and corresponding dorsal root ganglia on the experimental and contralateral side were dissected free and proceeded to AQP1 immunohistochemistry. Results Our quantitative estimations revealed that a sharp increase in AQP1 immunoreactivity at the 24th postoperative hour was observed. This sharp increase was no more evident at 48 h after sciatic nerve injury. Identical peak was observed after both cut and crush injuries. - The aquaporins family comprise a large family of integral membrane proteins that enable the movement of water and other small, neutral solutes across plasma membranes [1]. The function of these proteins is to control particular aspects of homeostasis [2,3]. The aquaporins (AQP) family in the central nervous system has diverse functions in neural signal transduction, cerebrospinal fluid formation, and osmoreception [4]. It has been reported that maintenance of osmotic composition and volume within the interstitial, glial, and neuronal compartments of the central nervous system are essential for normal function. Even small changes in osmolarity or volume can dramatically alter neuronal signaling and information processing [5,6]. It has been documented in the current literature that AQP1, AQP4, and AQP9 play important roles in brain water homeostasis [710]. Gao et al. [11] showed that AQP1 is main water channel in the peripheral nervous system. In the peripheral nervous system, AQP1 has been found to be expressed in trigeminal ganglion cells, periodontal cells, Ruffini endings, glial cells, dorsal root ganglia, terminal Schwann cells, nodose ganglion cells, and the enteric nervous system [10,1214]. Although function and mechanism of aquaporins in central nervous system injury have been reported, the pathophysiologic role of AQP1 in peripheral nerve has not been extensively documented. Therefore, further investigations in wellknown traumatic injuries of the peripheral nervous system are required to understand the temporal and spatial distribution of AQP1 in spinal cord and dorsal root ganglia and possible role (s) of AQP1 in intricate processes of the peripheral nerve injury/repair. Material and method Number, strain, and sex of animals Forty-eight adult female BalbC mice were obtained from the Laboratory Animal Unit of the Akdeniz University. Animals were housed in cages under standard environmental conditions (light between 06:00 and 18:00 h, temperature at 22 C, and free access to chow and water). All experimental protocols were approved by the Animal Welfare Committee of the Akdeniz University (protocol number 2013.05.04) and conducted in accordance with Turkish Law on the Protection of Animals. Animal groups and experimental design Study groups are depicted in Table 1. Throughout the study, allocation was concealed, i.e., the person undertaking the surgery did not know to which group the animal would be allocated. Animals were randomized into groups using a randomized number sequence and assessment was blinded. Group 1 (intact control, n= 6). In these animals, no surgical procedures were performed. They were intact mice. Group 2 (sham-operated, n= 6). Following sufficient anesthesia, skin of the lateral surface of the left thigh was incised in this group. Left sciatic nerve was exposed by a hind limb muscle splitting approach. After viewing the sciatic nerve, skin was immediately sutured with a 5.0 Ethilon suture and mice were allowed to recover in a postoperative room. Group 3 (cut injury, n= 18). In these animals, cut injury was performed. The animals were then randomized into groups using a randomized number sequence. Groups 3a, 3b, and 3c were sacrificed at the end of the 2nd, 24th, and 48th postoperative hours, respectively (Table 1). Group 4 (crush injury, n= 18). In these animals, cut injury was performed. The animals were then randomized into groups using a randomized number sequence. Groups 4a, 4b, and 4c were sacrificed at the end of the 2nd, 24th, and 48th postoperative hours, respectively (Table 1). Table 1 Study groups Group 4 (crush injury) After being prepared for aseptic surgery, animals were anesthetized with a mixture of xylazin HCI (15 mg/kg, Alfazyne, Alfasan International B.V., Woerden, Holland) and ketamine (100 mg/kg, Ketasol, Richter Pharma AG, Wels, Austria) via intramuscular injection. Following sufficient anesthesia, skin of the lateral surface of the left thigh was incised. A hind limb muscle splitting approach was used to expose the left sciatic nerve and its three branches under magnification with a fiberoptic-illuminated operating microscope (Olympus SZ61). Careful blunt dissection over a length of 1 to 1.5 cm was performed to isolate the sciatic nerve from the surrounding connective tissue. The nerve was cut transversely at the level of midthigh and repaired immediately by three to four stitches with 10.0 suture material (Fig. 1). Following the nerve cut and suture, muscle and skin layers were immediately sutured with a 5.0 Ethilon suture and mice were allowed to recover in a postoperative room. At the end of postoperative period (Table 1), animals were perfused (0.1 M phosphate buffered saline, pH 7.4; PBS) and fixed (4 % paraformaldehyde in 0.1 M phosphate-buffered saline, pH 7.4) transcardially. Following perfusion-fixation procedure, spinal cord samples at the level of lumbosacral intumescences and corresponding dorsal root ganglia on the experimental and contralateral side were dissected free and kept in 4 % paraformaldehyde in 0.1 M phosphate buffered saline, pH 7.4 (in dark and +4 C). All samples were postfixed in 4 % paraformaldehyde overnight and cryoprotected in 20 % sucrose in PBS. Transverse sections (30 m thick for spinal cord, 57 m thick for dorsal Fig. 1 AQP1 imm (...truncated)