A new model of experimental hemispherotomy in young adult Rattus norvegicus: a neural tract tracing and SPECT in vivo study

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OBJECTIVE

The objective of this study was to describe a new experimental model of hemispherotomy performed on laboratory animals.

METHODS

Twenty-six male young adult Wistar rats were distributed into two groups (surgery and control). The nonfluorescent anterograde neurotracer biotinylated dextran amine (BDA; 10,000 MW) was microinjected into the motor cortex area (M1) according to The Rat Brain in Stereotaxic Coordinates atlas to identify pathways and fibers disconnected after the experimental hemispherectomy. SPECT tomographic images of 99mTc hexamethylpropyleneamine oxime were obtained to verify perfusion in functioning areas of the disconnected and intact brain. A reproducible and validated surgical procedure is described in detail, including exact measurements and anatomical relationships. An additional 30 rodents (n = 10 rats per group) were divided into naïve, sham, and hemispherotomy groups and underwent the rotarod test.

RESULTS

Cortico-cortical neural pathways were identified crossing the midline and contacting neuronal perikarya in the contralateral brain hemisphere in controls, but not in animals undergoing hemispherotomy. There was an absence of perfusion in the left side of the brain of the animals undergoing hemispherotomy. Motor performance was significantly affected by brain injuries, increasing the number of attempts to maintain balance on the moving cylinder in the rotarod test at 10 and 30 days after the hemispherotomy, with a tendency to minimize the motor performance deficit over time.

CONCLUSIONS

The present findings show that the technique reproduced neural disconnection with minimal resection of brain parenchyma in young adult rats, thereby duplicating the hemispherotomy procedures in human patients.

ABBREVIATIONS BDA = biotinylated dextran amine; CPu = caudatus/putamen; HMPAO = hexamethylpropyleneamine oxime.

Article Information

Correspondence Norberto Cysne Coimbra: Ribeirão Preto Medical School of the University of São Paulo, Brazil. nccoimbr@fmrp.usp.br.

INCLUDE WHEN CITING Published online June 8, 2018; DOI: 10.3171/2017.12.JNS171150.

Disclosures Dr. Matias reports a financial relationship with Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq; National Council for Scientific and Technological Development), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; São Paulo Research Foundation), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; Coordination for the Improvement of Higher Education Personnel).

© AANS, except where prohibited by US copyright law.

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Figures

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    A: Flowchart showing the experimental design of the present work. Rats in the control group did not undergo neurosurgery. However, they were evaluated through neural tract tracing and neuroimaging procedures. Surgical intervention corresponds to the animals that underwent experimental hemispherotomy (EH). The EH group was initially composed of 21 animals: 8 died, 10 underwent neural tract tracing, and 3 underwent SPECT. B: Timeline of neuroanatomo-physiological investigation: 12 hours after neural tract tracing the animals were submitted to EH. The follow-up of the clinical evaluation occurred during 10 days, and the acquisition of SPECT images was performed 2 weeks after EH. BDA staining = visualization of interhemispheric neuroconnections; Histology 1 = tissue preparation (brain): perfusion, diaphanization, and paraffin fixation; Histology 2 = brain sectioning for the histochemical procedure. C: Timeline of the rotarod test procedure: habituation in the FMRP-USP (Faculdade de Medicina de Ribeirão Preto–University of São Paulo) Department of Surgery and Anatomy animal facility for 3 days, rotarod test before surgery on the 4th day, experimental hemispherotomy or sham procedure, and rotarod test 10 and 30 days after surgery.

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    Steps of the neurosurgical procedure (A–F). A: Sagittal encephalic view: disconnection of the frontal lobe and anterior commissure (arrowheads). B: Sagittal view: removal of cortex and subcortical white matter tissue block (dashed line). C: Callosotomy: corpus callosum and anterior commissure disconnection (arrowhead and dashed line). D: Sagittal and posterior coronal section of the brain, showing dorsal hippocampectomy (dashed line). E: Disconnection of external capsule (dashed line). F: Anteroposterior leucotomy from the amygdaloid complex to the occipital lobe. Parasagittal (G and I) and transverse (H, J, and K) sections of the rat encephalon showing representative histological views of the following surgical procedures: frontal disconnection and cortical resection (G); cortical resection with hippocampal preservation (H); calosotomy (I); cortical resection, dorsal hippocampectomy, and disconnection of the external capsule (J); and posterior leucotomy (K). Bars = 1000 μm.

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    Schematic view of the neuroanatomical procedure (neural tract tracing). A: Upper shows BDA anterograde neurotracer microinjections (arrowhead) in Rattus norvegicus M1 area of the neocortex (right hemisphere). Lower shows transverse section of Wistar rat brain demonstrating, in a schematic view, reciprocated neural pathways connecting both brain hemispheres through the corpus callosum. The photomicrograph on the left represents a histologically confirmed site of BDA microinjection in the telencephalon of the Wistar rat. B: Photomicrograph of a coronal section of the telencephalon of the Wistar rat showing a histologically confirmed site of intratelencephalic microinjection of the anterograde BDA neurotracer in the M1 cortical area. C: Representation of histologically confirmed sites of BDA microinjections in rat telencephalon of control and hemispherotomy-treated animals depicted in modified diagrams from the rat stereotactic atlas of Paxinos and Watson.

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    Photomicrographs of coronal sections of the telencephalon of a representative control Wistar rat, showing cortico-cortical pathways. A: Cortical fugal pathways (arrow) originated from the right brain hemisphere ipsilateral to the site of microinjection of the anterograde BDA neurotracer in the M1 cortical area, also sending inputs to the ipsilateral neostriatum (arrowheads). B: Intertelencephalic cortical pathways (arrowheads) crossing the midline in the corpus callosum (CC). C: Enhanced cortical fugal pathways (arrowheads) originating from the right brain hemisphere ipsilateral to the site of microinjection of BDA in the motor cortical area crossing the midline in the corpus callosum. D: Coronal sections of the left brain hemisphere showing BDA-labeled fibers (arrowheads) reaching the multiform cortical layer contralateral to the site of microinjections of the anterograde neurotracer, surrounding local neurons.

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    Coronal brain sections of a representative hemispherotomy-treated Wistar rat. A: Intrahemispheric cortico-neostriatal connections (arrowheads) and cortico-cortical pathways (arrow) originating from the right brain hemisphere ipsilateral to the microinjection site of anterograde BDA neurotracer in the M1 cortical area. B: BDA-labeled interhemispheric neural pathways (arrow) reaching the corpus callosum ipsilateral to the microinjection site performed in the M1 cortical area. C: Rarefaction of BDA-labeled fibers reaching the site of disconnection (arrowheads) of the corpus callosum near the midline. D: Note the lack of either BDA-labeled axonal fibers or neuronal perikarya in the contralateral cerebral cortex layers.

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    Coronal encephalic sections of a representative hemispherotomy-treated Wistar rat, showing preserved ipsilateral connections. A–C: The cortico-neostriatal pathway, with neurons (black arrows) situated in the internal pyramidal layer (A and B) sending axons (arrowheads) to the CPu (C). D: The neostriato-nigral GABAergic projection, represented by axons and terminal buttons (arrowheads) situated in the substantia nigra pars reticulata (SNpr). E and F: The nigro-striatal dopaminergic pathway, represented by neurons (arrows) situated in the substantia nigra pars compacta (SNpc) connected to the CPu (black arrowheads indicate neurites). The arrow in C shows a representative site of the BDA bidirectional neurotracer (BDA, 3000 MW) microinjections in the neostriatum. The neuronal body (arrow) shown at the bottom of panel D is situated in the SNpc. Axons (black arrowheads) and puncta (open arrowheads) suggesting terminal buttons shown in panel F on the right are situated in the SNpr. Counterstaining was conducted using Nissl cresyl violet.

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    SPECT images of the brain blood perfusion tracer 99mTc HMPAO in 1 normal and 3 hemispherotomy-treated rats. A: Representative horizontal and coronal sections through the brain images of the normal and one of the hemispherotomy-treated animals. B–E: Horizontal sequence of coronal sections through the brain images of the normal and 3 hemispherotomy-treated animals.

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    Effects of the hemispherotomy on motor performance of Wistar rats in the rotarod test during a 30-day follow-up. Data are presented as mean ± standard error of the mean (n = 10 per group). *p < 0.05, **p < 0.01 compared with the control group (naïve rats); #p < 0.05, ##p < 0.01 compared with the sham procedure group, according to the repeated 2-way ANOVA, followed by Bonferroni’s post hoc test. PreOp = preoperative/baseline responses.

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