ACE2 expression in rat brain: implications for COVID-19 associated neurological manifestations
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Abstract
We examined cell type-specific expression and distribution of rat brain angiotensin converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, in rodent brain. ACE2 is ubiquitously present in brain vasculature, with the highest density of ACE2 expressing capillaries found in the olfactory bulb, the hypothalamic paraventricular, supraoptic and mammillary nuclei, the midbrain substantia nigra and ventral tegmental area, and the hindbrain pontine nucleus, pre-Bötzinger complex, and nucleus of tractus solitarius . ACE2 was expressed in astrocytes and astrocytic foot processes, pericytes and endothelial cells, key components of the blood-brain-barrier. We found discrete neuronal groups immunopositive for ACE2 in brainstem respiratory rhythm generating centers including the pontine nucleus, the parafascicular/retrotrapezoid nucleus, the parabrachial nucleus, the Bötzinger and pre-Bötzinger complex and the nucleus of tractus solitarius; in arousal-related pontine reticular nucleus and in gigantocellular reticular nuclei; in brainstem aminergic nuclei, including substantia nigra, ventral tegmental area, dorsal raphe, and locus coeruleus; in the epithalamic habenula, hypothalamic paraventricular and suprammamillary nuclei; and in the hippocampus. Identification of ACE2-expressing neurons in rat brain within well-established functional circuits facilitates prediction of possible neurological manifestations of brain ACE2 dysregulation during and after COVID-19 infection.
Highlights
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ACE2 is present in astrocytes, pericytes, and endothelia of the blood brain barrier.
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Neuronal ACE2 expression is shown in discrete nuclei through the brain.
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Brainstem breathing, arousal-related, hypothalamic and limbic nuclei express ACE2.
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ACE2 is expressed in circuits potentially involved in COVID-19 pathophysiology.
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SciScore for 10.1101/2021.05.01.442293: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Euthanasia Agents: For immunohistochemistry, rats were deeply anesthetized with pentobarbital 63 mg/kg; when eyelid reflex was abolished, the animals were perfused transcardially with 0.9% NaCl solution followed by 0.1 M phosphate buffer (PB, pH 7.4) containing 4% paraformaldehyde and 15% v/v of a saturated picric acid solution.
IRB: Experimental protocols were reviewed and approved by the local Research and Ethics Committee (CIEFM-062-2016).Sex as a biological variable Animals and brain section preparation: Four male Wistar rats from the local animal breeding facility were used in the present study. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
… SciScore for 10.1101/2021.05.01.442293: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Euthanasia Agents: For immunohistochemistry, rats were deeply anesthetized with pentobarbital 63 mg/kg; when eyelid reflex was abolished, the animals were perfused transcardially with 0.9% NaCl solution followed by 0.1 M phosphate buffer (PB, pH 7.4) containing 4% paraformaldehyde and 15% v/v of a saturated picric acid solution.
IRB: Experimental protocols were reviewed and approved by the local Research and Ethics Committee (CIEFM-062-2016).Sex as a biological variable Animals and brain section preparation: Four male Wistar rats from the local animal breeding facility were used in the present study. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Antibodies Sentences Resources Primary antibody rabbit anti-ACE2 (ab15348, 1:1000, Abcam, MA, USA, see also table 1 for antibody information) was used, diluted in TBST + 1% NDS during 48h at 4°C with gentle shaking. anti-ACE2suggested: (Abcam Cat# ab15348, RRID:AB_301861)ab15348suggested: (Millipore Cat# AB15348, RRID:AB_805291)After this blocking step, sections were incubated during 48hr at 4°C with cocktails of primary antibodies against ACE2 and combinations of the following markers: calretinin (CR, a calcium binding protein with a high expression in several nuclei of the brainstem and in the olfactory bulb); the glycine transporter 2 (GlyT2, a marker of glycinergic neurons, highly expressed in the pontomedullary region of the brain); tyrosine hydroxylase (TH, a marker for catecholaminergic neurons), tryptophan hydroxylase (TPH, a marker for serotoninergic neurons); AVP-neurophysin (a marker for vasopressinergic neurons); neuron-specific nuclear protein (NeuN, a pan-neuronal marker); Von Willebrand Factor (VWF, a marker for endothelial cells) and glial fibrillary acidic protein (GFAP, a marker for astrocytes). calretinin ( CRsuggested: Noneglycine transporter 2suggested: NoneGlyT2suggested: Nonetyrosine hydroxylasesuggested: Nonetryptophan hydroxylase ( TPHsuggested: (GeneTex Cat# GTX61516, RRID:AB_10621478)neuron-specific nuclear proteinsuggested: NoneNeuNsuggested: Noneglial fibrillary acidic proteinsuggested: NoneGFAPsuggested: NoneExperimental Models: Organisms/Strains Sentences Resources Animals and brain section preparation: Four male Wistar rats from the local animal breeding facility were used in the present study. Wistarsuggested: RRID:MGI:5657554)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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