Male Sprague-Dawley rats weighing 270 to 330 gm were used for our studies. Rats were anesthetized with gas inhalation composed of 30% oxygen (0.3 L/ min) and 70% nitrous oxide (0.7 L/min) mixture. The gas was passed through an isoflurane vaporizer set to deliver 3% to 4% isoflurane during initial induction and 1.5% to 2% during surgery. An incision of the skin was made on top of the right common carotid artery region. The fascia was then blunt dissected until the bifurcation of the external carotid artery and internal carotidartery was isolated. A small incision was made on the external carotid artery, and a 19 to 22 mm long segment (depending on the weight of the animals) of 3–0 monofilament Nylon suture with a round tip was threaded into the internal carotid artery via the external carotid artery. The suture was advanced toward the middle cerebral artery (MCA) region to create focal ischemia. The suture was maintained in the vessel for 90 min then removed for reperfusion. Body temperature was maintained with a heating pad during surgery and during recovery from anesthesia.

A volume of 1 ml solution per 100 g body weight was given intravenously to all animals. Three solutions used in the study were: minocycline 1 mg/ml in saline, minocycline 0.3 mg/ml in saline, and saline alone. The corresponding dosages were 10 mg/kg or 3 mg/kg minocycline hydrochloride (Sigma, USA) or saline alone respectively. The solutions were administered and repeated slowly through jugular vein injection (lasting for 5 min) at three different administration protocols after TMCAO: for the 4 hour post TMCAO protocol 3 mg/kg or 10 mg/kg was given at 4, 8, and 12 hr; for the 5 hour post-TMCAO protocol at 5, 9, and 13 hr; and for the 6 hour post TMCAO protocol at 6, 10, and 14 hr.

Intensive monitoring of hemodynamic parameters was measured in animals undergoing TMCAO. The arterial blood pressure and heart rate were measured through a right femoral artery PE50 tubing using a PowerLab/400 data acquisition system and analyzed using PowerLab software (ADInstruments, CO, USA) according to the manufacturer's specification.

Femoral arterial blood samples of 0.3 ml each were directly drawn into a 1 ml syringe with a #25 gauge needle and analyzed for pH, oxygen (pO2) and carbon dioxide (pCO2) using a Roche OPTI CCA E-Glu Cassette (Roche Diagnostics Corporation, IN, USA) and an OPTI Critical Care Analyzer (AVL Scientific Corporation, Georgia, USA). These parameters and rectal temperature (using Homeothermic Blanket Systems, Harvard Apparatus, MA, USA) were studied at 10 min before TMCAO, 90 min, 240 min, and 360 min after reperfusion in rats with 90 min TMCAO and Minocycline (10 mg/kg, IV, at 4 hr after TMCAO) or saline (3 ml, iv, at 4 hr after TMCAO) treatments.

In 18 animals that did not undergo TMCAO, (6 animals received either 3, 10, or 20 mg/kg of minocycline intravenously), blood samples (1 mL) were drawn from the jugular vein at 30 minutes, 60 minutes, 120 minutes and 180 minutes after minocycline administration for assessment of maximum serum concentration (Cmax). After the samples were allowed to clot, serum was aspirated and frozen at -20 degrees Celsius until assayed.

Minocycline was assayed using a reverse-phase high performance liquid chromatographic method based on that developed by Orti et al15. Sensitivity of the method was 1 mg/L. Standard curves were linear over the range of 1 to 75 mg/L.

Infarct volume was measured using 2,3,5-triphenyltetrazolium chloride (TTC) stained brain slices (24 hr after TMCAO). This method provides an overall measure of cell injury presented by depleted NADPH and hence the inability to reduce TTC to its colored form.

Neurological deficits were examined at 24 hr after TMCAO just prior to sacrificing the animals using a 5-point scale adapted and modified from Zhang et al 16. Specifically, no neurological deficit, 0 point; right Horner's syndrome, failure to extend left forelimb, hindlimb, turning to left and circling to left, each scored 1 point.

Two-group, repeated measures ANOVA were used to examine differences in seven physiologic measurements: blood pressure, heart rate, temperature, pH, PO2, PCO2, and glucose. Six animals were used in each of two treatment groups: Minocycline or saline at 4 hours following TMCAO. Measurements were made for each physiologic parameter at 10 minutes before TMCAO, and 90, 240 and 360 minutes following reperfusion. In each ANOVA model, animal nested within treatment group was considered a random effect. Fixed effects included the treatment group and time of measurement. As well, the two-factor interaction between treatment group and time was included in each model.

Two-factor ANOVA was used to examine differences between treatment groups and administration times for the proportion infarct size and neurological score. In each model, treatment group (3 mg/kg minocycline, 10 mg/kg minocycline, or saline) and administration time (4, 5, or 6 hours) follow TMCAO were considered as fixed effects. The two-factor interaction between treatment group and administration time was also included in each model. All statistical significance was assessed using an alpha level of 0.05. Post hoc tests comparing the 3 mg/kg or 10 mg/kg to saline within each administration time (i.e. 6 comparisons) were conducted using a Bonferroni correction to the overall alpha level.

A linear relationship was observed between peak concentration and dose over the dose range studied (r = 0.998, Figure 1). Peak concentrations averaged 3.6, 13.0 and 28.8 mg/L with 3, 10 and 20 mg/kg doses respectively. The serum levels of MC at a 3 mg/kg dose (3.6 mg/L) were similar to that reported in humans after a standard 200 mg dose 14.

The results for the repeated measures ANOVA model for each outcome are given in Table 1. For each outcome, differences between treatment groups within time period and differences between time period within treatment group were examined using the adjusted least square means of the two-factor interaction between treatment group and time. There were no statistically significant differences between the minocycline or saline groups within any time point for any of the outcome measures: blood pressure, heart rate, temperature, pH, PO2, PCO2, or glucose.

Minocycline at doses of 3 mg/kg and 10 mg/kg IV was effective at reducing infarct size at 4 hours post TMCAO (figure 2). At doses of 3 mg/kg, minocycline reduced infarct size by 42% while 10 mg/kg reduced infarct size by 56% at 24 hours. Minocycline at a dose of 10 mg/kg significantly reduced infarct size at 5 hours by 40% and the 3 mg/kg dose significantly reduced infarct size by 34%. With a 6 hour time window there was a non-significant trend in infarct reduction (figure 2).

Examination of neurological function indicated that there was a significant amelioration of neurological deficits in animals that received 3 mg/kg or 10 mg/kg Iv minocycline at 4 hours post TMCAO compared to animals that received saline placebo. A signficant improvement in neurological scores was evident in the 10 mg/kg but not the 3 mg/kg dose compared to placebo at 5 hours. No significant differences in the neurological scores were seen at the 6 hour time windows (figure 3).