Introduction: Hydrocephalus is a brain disorder in which circulation of the cerebrospinal fluid (CSF) is impaired in such a way that CSF accumulates in its natural intracranial compartments (Curzio et. al., 2016). It is a serious problem in sub-Saharan Africa (Salvador et. al., 2014). It affects 1 to 3 of 1000 children at birth and is the most common neurologic disorder requiring surgery in children (Vogel et al., 2012). Stretching of the ependymal layer, thinning of the corpus callosum, extracellular oedema and reduced cortical thickness has been observed as the degree of hydrocephalus increased (Olopade et. al., 2012) as well as myelin sheath injury consisting of attenuation, lamella separation and accumulation of myelin debris and focal degeneration (Ayannuga et. al., 2016).
Pyramidal cells are the most abundant output neurons in the cerebral cortex (Rojo et. al., 2016). They are the major source of intrinsic excitatory cortical synapses and their dendritic spines are the main postsynaptic target of excitatory synapses (Elston and
Synapses develop concurrently and at identical rates in different layers of the cerebral cortex. They establish the cell-to-cell communication which is paramount to the functional capabilities of the cerebral cortex. Refinement of synaptic connections is a key step in the formation of neuronal circuits (Wang et. al., 2011). Synaptic vesicle membrane proteins participate in the storage of neurotransmitters, in the docking, fusion and recycling of synaptic vesicles (Grabs et. al., 1994).
Cortical areas do not possess most of their defining characteristics until post-natal time points (Stocker and O’Leary, 2016). There is a dearth of information on how the complex and orderly proliferation, lamination, synaptic transmission and most especially dendritic arborisation of the pyramidal neurons are affected by the hydrocephalic process.
Research questions
- Is the lamina architecture of the pyramidal neurons of layers 2, 3 & 5 of the sensorimotor cortex altered by hydrocephalus in neonatal mice?
- Does hydrocephalus affect dendritic arborisation of the pyramidal neurons layers 2, 3 & 5 of the sensorimotor cortex in neonatal mice?
- Does hydrocephalus affect the pre or post-synaptic structure in the sensorimotor cortex of neonatal mice?
Materials and Methods: An animal model of hydrocephalus has been set up in the Department of Anatomy of the University of Ibadan. Hydrocephalus will be induced in 15 neonatal (day-old) albino mice by intracisternal injection of 0.1 ml of 250 mg/ml sterile kaolin suspension. Control mice will undergo sham injection, but nothing injected into the cisterna magna will serve as controls. The animals will be weighed weekly and assessed for the development of hydrocephalus. These mice will be sacrificed on postnatal days 7, 14 and 21 by transcardial infusion of 4% paraformaldehyde in 0.1M phosphate buffer and their brains removed and stored in the same solution. The brains will be dissected out and post-fixed for 72 hours in the same solution. The brain will be bisected in the coronal plane, at right angle to a horizontal tangent at the level of the optic chiasm. The surface of the distal half of the brain so obtained will be examined grossly for the extent of ventricular dilatation.
Immunohistochemistry: Silver impregnation of fix brain tissue blocks will be done using a modified Golgi staining technique using 3% potassium dichromate and 2% silver nitrate solutions to evaluate the dendritic arborization of the pyramidal neurons as they extend across the vertical rows in the sensorimotor cortex.
Immunolabelling with Synaptophysin will also be carried out to assess immunoreactivity of the pre- and post-synaptic vesicles for the pyramidal neuronal maturation through differential expression in layers 2,3,5 of the sensorimotor cortex. The immunofluorescence labelled sections will be viewed with a light microscope, co-localisation of the cell types evaluated and photomicrographs taken. Cell counts will be done for both single and double labelled sections means ± standard errors of means (SEM) will generated and compared across the groups with confidence interval set at 95%. The impact of hydrocephalic process will be evaluated by comparing the number, size and synaptic morphology of the pyramidal neurons.
References
- Domenico L. Di Curzio, Emily Turner-Brannen, Xiaoyan Mao, Marc R. Del Bigio (2016). Magnesium sulfate treatment for juvenile ferrets following induction of hydrocephalus with kaolin. Fluids Barriers CNS; 13: 7.
- Elston G. N., Benavides-Piccini R., Elston A., Rimange P., DeFelipe J. (2011). Pyramidal cell in prefrontal cortex of primates: marked differences in neuronal structure among species. Neuroanat. 5:2.
- Elston and Manger P. (2014). Pyramidal cells in V1 of African rodents are bigger, more branched and more spiny than those in primates. Front Neuroanat; 8: 4.
- Hao Wang, Hong Liu, and Zhong-wei Zhang (2011). Elimination of Redundant Synaptic Inputs in the Absence of Synaptic Strengthening. The Journal of Neuroscience, 31(46):16675–16684
- Rojo C., Leguey I., Kastanauskaite A., Bielza C., Larrañaga P., DeFelipe J., Benavides-Piccione R. (2016). Laminar Differences in Dendritic Structure of Pyramidal Neurons in the Juvenile rat Somatosensory Cortex. Cerebral Cortex; 1–12.
- Vogel P., Read RW, Hansen GM, Payne BJ, Small D, Sands AT and Zambrowicz BP. (2012). Congenital Hydrocephalus in Genetically Engineered Mice. Veterinary Pathology 49(1) 166-181
- Stocker A.M., O’Leary D.D.M. (2016) Emx1 Is Required for Neocortical Area Patterning. PLoS ONE 11(2): e0149900. doi:10.1371/journal.pone.0149900
- Olopade FE, Shokunbi MT, Siren AL. (2012) The relationship between ventricular dilatation, neuropathological and neurobehavioural changes in hydrocephalic rats. Fluids Barriers CNS Sep 1:9(1):19
- Ayannuga O.A., Shokunbi M.T., Naicker T.A. (2016). Myelin Sheath Injury in Kaolin-Induced Hydrocephalus: A Light and Electron Microscopy Study. Pediatr Neurosurg; 51: 61-68
