Minocycline treatment following hypoxic/ischaemic injury attenuates white matter injury in a rodent model of periventricular leucomalacia

Abstract

Aims: Periventricular white matter injury in premature infants occurs following hypoxia/ischaemia and systemic infection, and results in hypomyelination, as well as neuromotor and cognitive deficits later in life. Inflammatory infiltrates are seen within human cerebral white matter from periventricular leucomalacia (PVL) cases.

Methods: In this study, we examine the time course of CD-68+ microglial cell responses relative to cell death within white matter following hypoxia/ischaemia in a rat model of PVL. We also tested the efficacy of the minocycline, an agent that suppresses microglial activation, in this model when administered as a post-insult treatment.

Results: We show that preoligodendrocyte injury in the post-natal day 6 begins within 24 h and continues for 48-96 h after hypoxia/ischaemia, and that microglial responses occur primarily over the first 96 h following hypoxia/ischaemia. Minocycline treatment over this 96 h time window following the insult resulted in significant protection against white matter injury, and this effect was concomitant with a reduction in CD-68+ microglial cell numbers.

Conclusions: These results suggest that anti-inflammatory treatments may represent a useful strategy in the treatment of PVL, where clinical conditions would favour a post-insult treatment strategy.

Figure 1

Temporal profile of cell death following unilateral hypoxic-ischaemic injury at post-natal day 6. (A) Highest levels of cells detected by in situ end labelling (ISEL) are seen at 24 and 48 h (upper middle and right: P7 and P8), decreasing by 96 h (lower middle: P10) following injury. One week (lower right: P14) after injury a rare ISEL+ cells were detected, a finding comparable to normal cell levels (upper left: Ctrl, ×1000 magnification). (B) Quantification of the time course of cell death following UCL/hypoxia at P6 (score: 0 ≤ 2 cells/field, 1 = 3–10 cells/field; 2 = 11–20 cells/field; 3 = 21–30 cells/field; 4 ≥ 31 cells/field). Normal control values at P6. (C) White matter pre-OL cell death 24 h post UCL/hypoxia at P6. TUNEL (red)/O4 (green) double staining. Scale bar = 20 μm. P, post-natal day; pre-OL, premyelinating oligodendrocyte; TUNEL, transferase-mediated dUTP nick end-labelling assay; UCL, unilateral carotid artery ligation.

Figure 2

Microglial infiltration in the white matter under (A) normal oxygenation and (B) after P6 UCL/hypoxia. Blinded cell counts CD-68+ cells in pericallosal white matter are shown as means ± SEM. CD-68+ cells are significantly increased ipsilateral to the lesion by 24 h and remain significantly increased for at least 96 h. CONTRA, contralateral; IPSI, ipsilateral; NML, normal; SEM, standard error of the mean; UCL, unilateral carotid artery ligation.

Figure 3

Effect of minocycline on the pericallosal white matter (WM) damage in P6 rats following UCL/hypoxia at 96 h. Myelin basic protein (MBP) staining shows less damage in minocycline-treated rats in comparison with vehicle-treated controls ipsilateral to UCL. (A) WM of vehicle-treated animal ipsilateral to UCL. (B) WM of vehicle-treated animal contralateral to UCL. (C) WM of minocycline-treated animal ipsilateral to UCL. (D) WM of minocycline-treated animal contralateral to UCL. (E) Quantification of WM injury using the lesion severity score (means ± SEM) based on MBP staining in minocycline-treated (n = 9) and vehicle (PBS)-treated animals (n = 9). Scale bar = 100 μm. Min, Minocycline; PBS, phosphate buffered saline; UCL, unilateral carotid artery ligation.

Figure 4

Effect of minocycline on the number of microglia in the pericallosal white matter (WM) in P6 rats following UCL/hypoxia at 96 h. CD-68 staining shows decreased number of CD-68+ cells in minocycline-treated rats in comparison with vehicle-treated controls ipsilateral to UCL. (A) WM of vehicle-treated animal ipsilateral to UCL. (B) WM of vehicle-treated animal contralateral to UCL. (C) WM of minocycline-treated animal ipsilateral to UCL. (D) WM of minocycline-treated animal contralateral to UCL. Scale bar, 100 μm. P6, post-natal day 6; PBS, phosphate buffered saline; UCL, unilateral carotid artery ligation.

Figure 5

Effect of minocycline on the number of CD-68-positive cells in the pericallosal white matter (WM) of P6 rats following UCL/hypoxia at 96 h. Results are shown as means ± SEM of CD-68+ count in brains of: minocycline-treated (n = 16) rats ipsilateral (IL-Minocycline) and contralateral (CL-Minocycline) to UCL; vehicle (PBS)-treated (n = 15) rats ipsilateral (IL-PBS) and contralateral (CL-PBS) to UCL; and healthy controls (n= 6). NS, not significant; P6, post-natal day 6; PBS, phosphate buffered saline; SEM, standard error of the mean; UCL, unilateral carotid artery ligation.

Figure 6

Effect of minocycline on the number of CD-74 (MHC II)-positive cells in the pericallosal white matter (WM) in P6 rats following UCL/hypoxia at 96 h. CD-74 staining shows decreased number of CD-74+ cells in minocycline-treated rats in comparison with vehicle-treated controls ipsilateral to UCL. (A) WM of vehicle-treated animal ipsilateral to UCL. (B) WM of vehicle-treated animal contralateral to UCL. (C) WM of minocycline-treated animal ipsilateral to UCL. (D) WM of minocycline-treated animal contralateral to UCL. Scale bar, 100 μm. MBP, myelin basic protein; P6, post-natal day 6; PBS, phosphate buffered saline; UCL, unilateral carotid artery ligation.