Esomeprazole’s Antifibrotic Effects on Rats With Epidural Fibrosis

Experimental Animals

This study, which obtained approval from the University of Health Sciences’ regional animal research ethics committee (20.11.2020, 06.09), adhered strictly to the European Union Council Directive (86/609/EEC) of November 24, 1986 addressing the protection of animals used in studies. The study followed all the directives of animal care and testing. A total of 18 female Sprague Dawley rats, each weighing between 150 and 250g, were the subjects of the experiment. They were housed under optimal conditions with a 12-hour light cycle, controlled humidity, and temperatures maintained between 22 and 25°C. They had unrestricted access to food and water throughout the experiment.

These animals were randomly divided into three groups for the study:

Surgical Procedure

To prevent infection, each rat received an intramuscular 20 mg/kg sodium cephazolin injection before surgery. Intraperitoneal injections of 50 mg/kg ketamine (Ketalar, Parke Davis, Turkey) and 10 mg/kg xylazine (Rompun, Bayer, Turkey) induced anesthesia. Rat body temperatures were maintained at 37°C using a heating pad and a rectal probe.

The rats were placed in a supine position during surgery. Their lower backs were shaved, the surgical sites were cleaned with povidone and a sterile field was established. A midline incision was made over the L1-L3 levels and laminectomy at the L2 level then exposed the dura mater and epidural spaces, with careful removal of the ligamentum flavum and epidural fat tissue. Topical treatments were applied in the relevant groups and the wounds were sutured with 4-0 prolene polypropylene sutures. The same surgeons meticulously carried out each surgery under a surgical microscope to protect the neurological structures (BG, PKB).

Post-surgery, the rats had unrestricted access to food and water for four weeks. Euthanasia was performed via cervical dislocation following an intraperitoneal injection of 50 mg/kg ketamine (Ketalar, Parke Davis, Turkey). The laminae, dural sacs, nerve roots, and paravertebral tissues, along with the muscles and skin, were excised from the first to the third vertebrae.

Macroscopic Assessment of Epidural Fibrosis

A neurological surgeon blind to the treatment groups carefully re-opened the surgical sites and macroscopically evaluated the existing epidural fibrosis according to the Rydell classification, ¹² which includes four distinct grades based on scar tissue adherence to the dura mater and the difficulty of dissection. The details of Rydell classification scoring were published recently. ¹³

Histopathological Evaluation

For histological analysis, the laminectomy region was separated by cutting the upper L1 to lower L3 levels of the spine axially. Pathology procedures were published previously. ¹³ For histological analysis, the samples were immersed in a 10% buffered formalin solution. The specimens were separated into 2-3 mm thick axial slices and then preserved in formalin for 48 hours. A decalcification solution (BiocalC, Code: RRDC3/G, Specifications: EDTA1%, Pot. Sod. Tartate1%, Sodium Tartate1%, Hydrochloric acid1%, Biostain-U.K.) was used to decalcify each tissue sample for 72 hours. Hematoxylin and eosin (H&E, Shandon Harris Hematoxylin-Eosin Y -UK) and Masson trichrome (Bio-Optica special stains kit, Milan, Italy) were used to stain serial slices that were four μm thick after a paraffin-embedded block was prepared from formalin-fixed, paraffin-embedded tissues. Bozkurt et al. ¹⁴ ’s parameters for the microscopic assessment of EF were used to assess the extent of inflammation, scar tissue formation, fibrosis, and other histological changes. The classification scheme for fibroblast cell density was published recently. ¹³

Statistical Analysis

GraphPad Prism 8.0 (GraphPad Software Inc, La Jolla, CA, USA) was used for data analysis. A one-way analysis of variance was used to examine the variations between the groups, followed by a Tukey posthoc test. The threshold for statistical significance was a P value of 0.05.

Wound Healing and Complications Related to the Procedure

In the local esomeprazole group, 2 unfortunate instances of mortality were recorded. As the dosage was arranged accordingly and even no slight systemic symptoms were observed in other animals in this group, we speculate that these instances may have been caused by animal-related reasons. The local effects of esomeprazole need further investigation to have a conclusion. There were no procedural complications such as hematomas, erythema, infections, or leaks of cerebrospinal fluid reported across all groups. At the time of sacrifice, all animals retained unhindered mobility.

Macroscopic Assessment of Epidural Fibrosis

Severe epidural adhesions were observed in the majority of cases in the laminectomy group (n = 5 grade 3 and n = 1 grade 2). Moderate to severe epidural adhesions were observed in the local esomeprazole group (n = 3 grade 3 and n = 1 grade 2). Only mild to moderate epidural adhesions were observed in the systemic esomeprazole group (n = 3 grade 1, n = 1 grade 2). Notably, the epidural fibrosis severity score was statistically significantly decreased in the systemic esomeprazole group compared to the laminectomy group (P < 0.001). However, no statistically significant difference was found in comparing the local esomeprazole group to the laminectomy group (Figure 1).OPEN IN VIEWER

Histopathologic Assessment

The microscopic evaluation of EF scores showed no statistically significant difference in the local and systemic esomeprazole groups compared to the laminectomy group. However, the EF grades for local and systemic esomeprazole groups were lower than the laminectomy group. The fibroblast cell density grades did not show a statistically significant decrease in both local and systemic esomeprazole groups compared to the laminectomy group. However, the mean fibrosis thickness showed a decrease in both the local and systemic esomeprazole groups when compared to the laminectomy group (P < 0.01, P < 0.001, respectively) (Figures 1 and 2, Table 1). Interestingly, cartilage destruction was noted in 2 rats in the local esomeprazole group, and bone destruction was reported in 1 rat in the same group. The responses to foreign bodies did not differ significantly among the groups. Mild chronic inflammation was more commonly seen in the laminectomy group when compared to local and systemic esomeprazole groups.

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Table 1.

Microscopic Evaluation of Experimental Groups.

	LAM	L-ESO	S-ESO
Epidural fibrosis grade	Grade 2: n = 1	Grade 2: n = 2	Grade 2: n = 4
	Grade 3: n = 5	Grade 3: n = 2	Grade 3: n = 2
Mean fibroblast cell density grade	Grade 2: n = 4	Grade 2: n = 4	Grade 2: n = 5
	Grade 3: n = 2		Grade 3: n = 1
Mean fibrosis thickness (mm)	2.82 ± 0.41	1.75 ± 0.29	1.56 ± 0.41
Cartilage destruction	None	n = 2	None
Bone destruction	None	n = 1	None
Foreign body reaction	n = 3	n = 3	n = 3
Mild chronic inflammation	n = 2	n = 1	n = 0