A New Precision Minimally Invasive Method of Glial Scar Simulation in the Rat Spinal Cord Using Cryoapplication

Abstract

According to the World Health Organization, every year worldwide up to 500,000 people suffer a spinal cord injury (SCI). Various animal biomodels are essential for searching for novel protocols and therapeutic approaches for SCI treatment. We have developed an original model of post-traumatic spinal cord glial scarring in rats through cryoapplication. With this method the low-temperature liquid nitrogen is used for the cryodestruction of the spinal cord tissue. Forty-five Sprague Dawley (SD) non-linear male rats of the Specific-pathogen-free (SPF) category were included in this experimental study. A Th13 unilateral hemilaminectomy was performed with dental burr using an operating microscope. A specifically designed cryogenic probe was applied to the spinal cord for one minute through the created bone defect. The animals were euthanized at different time points ranging from 1 to 60 days after cold-induced injury. Their Th12-L1 vertebrae with the injured spinal cord region were removed “en bloc” for histological examination. Our data demonstrate that cryoapplication producing a topical cooling around−20°C, caused a highly standardized transmural lesion of the spinal cord in the dorsoventral direction. The lesion had an “hour-glass” shape on histological sections. During the entire study period (days 1-60 of the post-trauma period), the necrotic processes and the development of the glial scar (lesion evolution) were contained in the surgically approached vertebral space (Th13). Unlike other known experimental methods of SCI simulation (compression, contusion, etc.), the proposed technique is characterized by minimal invasiveness, high precision, and reproducibility. Also, histological findings, lesion size, and postoperative clinical course varied only slightly between different animals. An original design of the cryoprobe used in the study played a primary role in the achieving of these results. The spinal cord lesion's detailed functional morphology is described at different time points (1–60 days) after the produced cryoinjury. Also, changes in the number of macrophages at distinct time points, neoangiogenesis and the formation of the glial scar's fibrous component, including morphodynamic characteristics of its evolution, are analyzed. The proposed method of cryoapplication for inducing reproducible glial scars could facilitate a better understanding of the self-recovery processes in the damaged spinal cord. It would be evidently helpful for finding innovative approaches to the SCI treatment.

Plasma Cytokines Level and Spinal Cord MRI Predict Clinical Outcome in a Rat Glial Scar Cryoinjury Model

“…We used a previously elaborated model of spinal cord cryoinjury [ 12 , 13 ] characterized by monoplegia of the hind limb, which can last several months ( Figure 1 A). Functional tests showed that intact animals easily reached the preset maximum speed of 50 m/min, whereas the animals with spinal cord injury reached a mean speed of 19 m/min.…”

“…In this study we used a recently proposed rat SCI model accomplished by a controlled cryoapplication. Our technique, unlike other models of traumatic spinal cord injury, consistently reproduces glial scarring and the posttraumatic formation of cysts [ 13 ], which both play a fundamentally negative role against potential neuroregeneration [ 15 , 16 , 17 ].…”

“…Histological studies of rat spinal cord were performed at days 1, 7, 14, 21, 30 and 60, as described previously [ 12 , 13 ]. Briefly, samples of the rat spinal cord encased in bone corresponding to the length of three vertebrae (Th13 was the vertebra of the surgical approach, and Th12 and L1 were the two adjacent vertebrae) were fixed in 10% neutral buffered formalin, rinsed in tap water and processed for decalcification in Trilon B at room temperature for 12–16 days.…”

“…Here, we used a previously described, highly reproducible rodent model of unilateral controlled spinal cord trauma induced by a cryoinjury [ 12 , 13 ]. We studied specifically the time course of the posttraumatic process with magnetic resonance imaging (MRI) and the concentration of cytokine plasma and colony-stimulating factors as related to SCI clinical outcome.…”

Refinement of the motorised laminectomy-assisted rat spinal cord injury model by analgesic treatment

“…Our study used a modified technique to perform laminectomy, using a motorised dental burr to mechanise the process to refine the process in terms of animal wellbeing, as previously described [ 25 , 85 ]. Even though refined techniques to mechanise laminectomy and reduce bleeding, inflammation and trauma has been adopted by certain researchers [ 86 , 87 ], a majority of publications on the induction of SCI applies the manual, conventional technique to perform laminectomy. So, it is required to replicate this study by performing laminectomy manually to study the effects of analgesia which is also being planned.…”