A rapid fluorescent method to quantify neuronal loss after experimental intracerebral hemorrhage

Abstract

Neuronal loss in tissue surrounding an intracerebral hemorrhage (ICH) is usually quantified by labor-intensive histological methods that are subject to bias. Fluorescent protein expression has been successfully used as a marker of cell viability in vitro and in retinal studies in vivo, but not in any ICH model to date. The potential of this approach was investigated using transgenic mice that constitutively express the red fluorescent protein variant dTomato in central neurons under the control of the Thy1 promoter. Breeding and growth of these mice were similar to their wild-type counterparts; behavioral phenotyping by digital analysis of home cage video recordings detected no differences. Bright fluorescence was evident in fresh brain samples with minimal background fluorescence, and was reduced in tissue surrounding the hematoma. In order to assess fluorescence loss as an injury marker in a planned study, these mice were crossed with heme oxygenase (HO)-2 knockouts and wild-type controls; striatal hemorrhage was induced by stereotactic injection of collagenase. Fluorescence in hemorrhagic striata was reduced to 86.4±3.9%, 62.2±5.1%, and 58.3±3.0% of contra-lateral on days 1, 4 and 8, respectively, and correlated closely with reduction in striatal cell viability as quantified by MTT assay. HO-2 knockout and wild-type values did not differ significantly. Similar results were observed with stereological cell counts of striatal neurons identified by NeuN immunoreactivity. These results suggest that loss of constitutive dTomato fluorescence is an accurate and efficient marker of neuronal loss in tissue surrounding a striatal hematoma.

Figure 1

A) Behavioral phenotyping by digital analysis of video recordings demonstrated no difference in cage activity of mice lacking the dTomato gene and wild-type at the HO-2 locus (WT), transgenic dTomato mice with the wild-type HO-2 gene (RFP-WT), and dTomato transgenic HO-2 knockout mice (RFP-KO). Total minutes spent walking, rearing, feeding, vertical hanging and jumping were summed to provide index of total activity; B) Representative gels identifying: top panel, HO-2 protein expression by western blotting; middle panel, HO-2 wild-type gene and knockout insert by PCR, control is from HO-2 heterozygous knockout mouse; bottom panel, Brainbow construct containing dTomato gene by PCR, control is from wild-type mouse without dTomato gene; C) Identification of dTomato expression in living mouse by skull transillumination after hair clipping (left 4 photos), and in freshly harvested brain (right 4 photos), using a TRITC filter. Scale bar = 3 mm.

Figure 2

Brightfield (top panel) and corresponding fluorescent (bottom panel) images of dTomato-expressing brain (wild-type at HO-2 locus), 4 days after injection of 0.028 units collagenase into the right striatum. A) whole brain before dissection; B) left striatum (contralateral to collagenase injection) is exposed by removal of overlying cortex, demonstrating bright fluorescence; C) injected right striatum is exposed, demonstrating reduced fluorescence in superior aspect of striatum; D) left and right striata have been removed; reduced fluorescence of hemorrhagic right striatum is apparent. Scale bar = 3mm.

Figure 3

Loss of dTomato fluorescence after striatal ICH correlates with attenuation of MTT reduction to formazan. Bars represent mean striatal cell formazan or striatal dTomato fluorescence 1, 4 or 8 days after stereotactic injection of 0.014 or 0.028 units collagenase (high dose). Controls were injected with an equal volume of artificial CSF instead of collagenase. All mice used expressed dTomato; WT and KO indicate gene at the HO-2 locus. Values are normalized to those in the contralateral striatum (= 100), n = 5–12 mice/condition.

Figure 4

dTomato fluorescence is reduced in the right striata of HO-2 WT and KO mice after collagenase-induced ICH. Serial sections (300 µm) were cut coronally through the striatum, 4 days after striatal injection of 0.028 units collagenase. Top row (WT) and third row (KO) are brightfield images; second row (WT) and last row (KO) are images under fluorescence microscope.

Figure 5

Correlation of dTomato fluorescence and MTT reduction to formazan in mice (n=52) receiving striatal injection of collagenase or artificial CSF.

Figure 6

HO-2 gene knockout does not alter striatal hemorrhage after collagenase-induced ICH. Mean striatal hemoglobin in HO-2 knockout and wild-type striata 24 hours after injection of 0.014 or 0.028 units collagenase (n=5/condition).

Figure 7

Bars represent mean number of striatal NeuN immunoreactive cells in HO-2 wild-type (WT) and knockout (KO) mice 4 days after injection of collagenase (0.028 units) or artificial CSF, and in contralateral striata. Total cell numbers are based on sampling using design-based stereology. Photographs are of representative WT and KO sections stained with anti-NeuN; C,G are from contralateral striata, D,H are from collagenase-injected striata. Note that stereological cell counting requires thick sections, so some neuron cell bodies are out of focus. Scale bar = 10 µm.

Figure 8

Effect of HO-2 knockout on behavioral outcome after ICH. Mouse cage activity and performance on adhesive removal, corner, and elevated body swing tests at indicated days after 0.014 unit collagenase injection (*P < 0.05 v. corresponding WT condition, Bonferroni multiple comparisons test, n = 6–22/condition, with higher value for pre-injection testing).