Effects of minocycline on epiplexus macrophage activation, choroid plexus injury and hydrocephalus development in spontaneous hypertensive rats

Abstract

Hydrocephalus has been reported to occur in spontaneous hypertensive rats (SHRs). The purposes of this study were (1) to use T2 magnetic resonance imaging to examine time of onset, (2) to elucidate potential underlying mechanisms and (3) to determine whether minocycline could prevent hydrocephalus development. Ventriculomegaly was evaluated by T2 imaging in SHRs and Wistar-Kyoto rats from weeks 4 to 7 after birth. Brain histology and transmission electron microscopy were used to assess the periventricular and choroid plexus damage. SHRs were also treated with either vehicle or minocycline. We found that hydrocephalus was observed in SHRs but not in Wistar-Kyoto rats. It occurred at seven weeks of age but was not present at four and five weeks. The hydrocephalus was associated with epiplexus cell (macrophage) activation, choroid plexus cell death and damage to the ventricle wall. Treatment with minocycline from week 5 attenuated hydrocephalus development and pathological changes in choroid plexus and ventricular wall at week 7. The current study found that spontaneous hydrocephalus arises at ∼7 weeks in male SHRs. The early development of hydrocephalus (persistent ventricular dilatation) may result from epiplexus cell activation, choroid plexus cell death and periventricular damage, which can be ameliorated by treatment with minocycline.

Keywords: Choroid plexus; epiplexus cells; hydrocephalus; minocycline; spontaneous hypertensive rats.

Figure 1.

Spontaneous hydrocephalus occurs in SHRs at seven weeks of age. (a) Representative T2-weighted MRI images showing ventricular enlargement in SHRs at week 7 compared with age-matched WKY rats. (b) Quantification of ventricular volume on T2 images at weeks 4, 5 and 7. Values are means ± SD, n = 6; #P < 0.01 compared with WKY group. (c) MRI T2 showing evidence of the aqueduct and 4th ventricular enlargement in SHRs at week 7. (d) SHRs had significant higher systolic blood pressure (SBP) and mean arterial pressure (MAP) than WKY rats at weeks 5, 6 and 7. #P < 0.01 compared with WKY group, n = 6.

Figure 2.

SHRs have evidence of epiplexus macrophage activation compared to WKY rats. (a) Examples of choroid plexus (CP) Iba-1 immunohistochemistry in SHRs and WKY rats at week 7 (low and high magnification). Note the presence of more and larger Iba-1-positive cells on the apical surface of the CP in the SHRs. Bar graphs show quantification of the number of Iba-1 cells (as a % of all cells) in both WKY rats and SHRs and measurement of the size of the macrophages. Values are means ± SD, #P < 0.01 compared with WKY group, n = 6. Scale bar = 50 μm (low magnification) and 10 μm (high magnification). (b) CD68-positive epiplexus macrophages in SHRs and WKY rats at week 7. Values are means ± SD, #P < 0.01 compared with WKY group, n = 6. Scale bar = 50 μm.

Figure 3.

Choroid plexus (CP) ultrastructural changes and CP cell death in SHRs and WKY rats at week 7. (a) H & E staining of WKY sections showed organized CP epithelial cells with homogeneous nuclei at week 7 (*), while CP cells were sometimes disoriented with atrophic nuclei (arrow) in SHRs. Endothelium of capillaries were intact and closely associated with the epithelium in WKY rats but sometimes fragmented in SHRs (star). (b) Microbleeding of CP in the SHR at week 7. There was evidence of erythrocytes leaking out of endothelium and epithelium into ventricle (arrow) and hematoceles (star). (c) TUNEL staining and quantification showed abundant TUNEL-positive cells in CP of SHRs at week 7. #P < 0.01 compared with WKY group, n = 6. Scale bar = 50 μm (low magnification) and 20 μm (high magnification). (d) CP ultrastructure in SHRs and WKY rats at seven weeks of age as assessed by transmission electron microscopy. Low-magnification image showing CP epithelia in WKY rats characterized by round nuclei and clear cell cytoplasm. In contrast, in SHRs, some epithelial cells had atrophied nuclei (arrow) surrounded with abnormal cytoplasm (star). In WKY rats, mitochondria were normal in shape and scale, while in SHRs, mitochondria appear slightly swollen (arrow) and in some cells were surrounded by multiple vesicles/cysts (star). LV: lateral ventricle; mvi: microvilli.

Figure 4.

Ventricular wall damage in SHR and WKY rats at weeks 4 and 7. (a) Hematoxylin and eosin- (H & E) stained sections were examined at three different layers of ventricular system for ventricular wall damage. Scale bar = 1 mm. (b) Typical ventricle wall injury including detachment of ependymal layer (white arrow), sparse white matter (arrowhead), bulge (star) and cells shedding (black arrow) was found in SHRs at week 7. Scale bar = 50 μm (high magnification) and 100 μm (low magnification). (c) Quantification of ventricle wall damage (percentage of total perimeter). SHRs had more damage on layer 2 at week 4 and on all the three layers at week 7. Values are means ± SD; *P < 0.05 compared with WKY group; n = 6.

Figure 5.

Minocycline reduced hydrocephalus and hypertension in SHRs at week 7. T2 images (a) and quantification of ventricular volume (b) at weeks 5 and 7 in animals treated with vehicle, 10 or 20 mg/kg minocycline (mino) from week 5. Both minocycline doses reduced the ventricular enlargement found at week 7. (c) The effects of minocycline on hydrocephalus were confirmed by examining coronal sections of frozen brains. Representative MRIs and frozen sections from the three groups. (d) Minocycline treatment also significantly reduced systolic and mean arterial pressures at weeks 6 and 7. Values are means ± SD, *P < 0.05 compared with SHR + vehicle group, #P < 0.01 compared with SHR + vehicle group; n = 9.

Figure 6.

Minocycline reduced epiplexus macrophage activation in SHRs. (a) Examples of choroid plexus (CP) Iba-1 immunohistochemistry (low and high magnification) in SHRs treated with 10 or 20 mg/kg minocycline (mino). Rats were treated for two weeks and examined at week 7. Note the reduction in the Iba-1 immunoreactivity in cells on the apical CP surface with minocycline treatment. Bar graphs showing quantification of the number of Iba-1 cells (as a % of all cells) with and without minocycline treatment and measurement of the size of the macrophages. Values are means ± SD, #P < 0.01 compared with SHR + Vehicle group, n = 9. Scale bar = 50 μm (low magnification) and 10 μm (high magnification). (b) CD68 immunohistochemistry in SHRs treated with 10 or 20 mg/kg minocycline (mino). Values are means ± SD, #P < 0.01 compared with SHR + Vehicle group, n = 9. Scale bar = 50 μm.

Figure 7.

Cell death in the choroid plexus and ventricular wall damage were alleviated by minocycline. (a) Examples of TUNEL staining in the choroid plexus in SHRs treated with vehicle or 10 or 20 mg/kg minocycline. Note the reduced prevalence of TUNEL-positive cells with minocycline treatment. Quantification of the number of TUNEL-positive cells in the choroid plexus (at level 1). Values are #P < 0.01 compared with SHR + vehicle group; n = 9. Scale bar = 50 μm (low magnification) and 20 μm (high magnification). (b) Quantification of ventricular damage in the three groups. Both minocycline groups had reduced ventricular wall damage compared to the vehicle group. Values are means ± SD, #P < 0.01 compared with SHR + vehicle group, n = 9.