Isoflurane

The three types of mice (Korl:ICR, A:ICR, and B:ICR) were each divided into two groups (negative control group and botulinum toxin-induced group; n = 10 in each group). Before the experiment, all mice were acclimatized for 7 days. Botulinum-toxin A (BoNT/A, Botox, 100 U, Allergan, Irvine, CA) was injected into the botulinum toxin-induced group mice and saline into the negative control group mice. The left hind limbs were shaved and a single injection of 1.0 U/kg BoNT/A into the gastrocnemius muscle. Body weight was measured daily for 3 days using an electrical balance (FX-2000i, AND, Japan). After the experiment, all mice were anesthetized using 2% isoflurane (Virbac, UK) in a chamber. Blood samples were collected from the abdominal aorta and stored into a BD Microtainer Blood Collection Tube (BD Life Sciences, Franklin Lakes, NJ, USA) for hematology and serum biochemistry analysis. The muscle tissues were extracted for histopathology analysis.

Neurotransmitter concentrations were measured in the frontal cortex, the main area of serotoninergic projections that exhibited the prominent number of spike-wave discharges after 10 to 40 mg/kg tramadol administration [63 (link)]. Following tramadol administration, rats were immediately placed for 10 min in an anesthesia induction chamber (Tem Sega, Pessac, France) with 4% isoflurane (Virbac, Carros, France). They were exsanguinated to clean the brain of its blood and then decapitated. The frontal cortexes were collected 15–20 min after drug administration. Samples were frozen at −80 °C until measurement. Concentrations of monoamines, i.e., dopamine, serotonin, norepinephrine and their metabolites, 5-hydroxyindoleacetic acid (5-HIAA), 3-methoxy-4-hydroxyphenoglycol (MHPG), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured using high-performance liquid chromatography (HPLC) coupled to flurorimetry as previously described [64 (link)]. Radioenzymatic assay was used for histamine concentration determination as previously described [65 (link)].

Following the experiment, all mice were anesthetized in a chamber using 2% isoflurane (Virbac, UK). Brain tissues were extracted for histopathology analysis and fixed with 10% neutral buffered formalin solution (BBC Biochemical, Mount Vernon, WA, USA) for 1 week. The fixed tissues were embedded in a paraffin block and sectioned into 4-μm thick sections. The sections were then mounted on slides and stained with hematoxylin and eosin (H&E) solution using an autostainer (Dako Coverstainner; Agilent, Santa Clara, CA, USA). Additionally, the tissue sections were stained immunohistochemically using the labeled polymer DAKO EnVisionTM+System-HRP (Agilent) according to the manufacturer’s instructions. The brain sections were stained with anti-GFAP (ab7260; Abcam, Cambridge, MA, USA), anti-Iba1 (ab5076; Abcam, Cambridge, MA, USA), and anti-NeuN (ab177487; Abcam, Cambridge, MA, USA) primary antibody. After staining, all areas of the brain that were scanned with a slide scanner (Pannoramic SCAN II; 3DHISTECH, Budapest, Hungary) and were captured by a slide viewer (CaseViewer; 3DHISTECH, Budapest, Hungary). For quantification analysis, Each slide captures 3 points at 400x in the hippocampus. Using image analyzer software Image J (NIH, MD, USA), the positive portion of the total is quantified as a percentage and the average is calculated.