Animals, hyperglycemia induction, and surgery

Hyperglycemia was induced using nicotinamide-streptozotocin. Streptozotocin has a nonspecific action in the body that causes organ deterioration due to its cytotoxic action [15, 16]. Nicotinamide was used to prevent organ damage [15]. In the present study, hyperglycemia was induced by an intraperitoneal injection with nicotinamide (210 mg/kg) (EMD Millipore, Germany) followed 15 min later with an intraperitoneal injection of streptozotocin (60 mg/kg) (EMD Millipore, Germany). After 72 h and again at 6 weeks, blood glucose concentration was determined using a glucose meter (ACCU-CHEK, Performa, Germany), and the animals with a blood glucose concentration above 250 mg/dl were considered hyperglycemic [17].

A total of 57 male Sprague-Dawley rats (8–9 weeks old, weighing 200–250 g) were used for the study. The rats were anesthetized via intraperitoneal injection of a solution of ketamine (25 mg/kg) and diazepam (2 mg/kg) at a dose of 2.5 ml/kg. Analgesia was induced by subcutaneous injection of meloxicam (2 mg/kg). To perform the pMCAO, a small craniotomy was performed; the right middle cerebral artery (MCA) was permanently ligated just before its bifurcation and both common carotid arteries were then occluded for 60 min as previously described [18].

The rats were randomly assigned to the following groups: the sham group was subjected to surgery without infarction (n = 10); the non-hyperglycemic group was subjected to a pMCAO and received intravenous saline (n = 10); the nontreated hyperglycemic group was subjected to a pMCAO and received intravenous saline (n = 10); and the treated hyperglycemic group was subjected to a pMCAO and received intravenous hAD-MSCs (n = 11). Saline solution or 1 × 10⁶ hAD-MSCs in 1 ml of saline solution was administered via the tail vein at 48 h after surgery. All the animals were euthanized at 6 weeks post-stroke (Fig. 1b).