Hydrocephalus is characterized by enlargement of the cerebrospinal fluid (CSF)-containing ventricles of the brain. Several factors including tissue compression, stretching of axons, ischemia, and calcium-mediated proteolysis simultaneously contribute to axon injury in periventricular white matter [
1,
2]. We have also postulated that movement and composition of extracellular fluids are altered with potential reversible effects on neuronal function [
3] and Hakim suggested that the brain behaves like a compressed sponge with reduction of the extracellular compartment [
4]. Although the periventricular white matter extracellular compartment is enlarged in progressive hydrocephalus [
3,
5], several types of evidence suggest that the more superficial parts of brain are indeed compressed. Tissue density measurements in adult rabbits with silicone oil-induced hydrocephalus indicated increased density (possibly decreased water content) in cortical gray matter [
6], although this was not replicated in a similar study that used wet-dry weight comparisons [
7]. Computed tomography scanning of human brains suggests that brain hydration is reduced when the ventricles expand [
8]. Freeze-substitution electron microscopic studies of hy-3 mutant mice show that the extracellular compartment in the superficial cerebral cortex is initially compressed and subsequently enlarged [
9]. Increased electrical impedance has been interpreted as a reflection of decreased water content in thalamus of hydrocephalic cats [
10]; similar findings have been obtained in human studies [
11,
12]. Apparent diffusion coefficient (ADC) mapping in rats with hydrocephalus suggest that the movement of water in rat brain is restricted in gray matter but not in white matter [
13]. Extracellular infusion of gadolinium-DTPA followed by repeated magnetic resonance (MR) imaging [
14] and real-time iontophoretic tetramethylammonium diffusion studies [
15] suggest that the extracellular flow and volume fraction is reduced in the cerebral cortex of rats with kaolin-induced hydrocephalus. In the H-Tx rat with congenital hydrocephalus, ionic composition studies suggest that the extracellular compartment in the cortex is enlarged 4–21 days after birth [
16] while iontophoretic studies indicate that the extracellular volume fraction is reduced [
15]. In conjunction with the obstruction to CSF outflow, composition of the CSF is altered [
17].
It is important to determine if this impairment of extracellular flow in hydrocephalic brains is purely due to physical compression, which could be reversible upon shunting, or if there are structural or compositional changes in the extracellular compartment. If the latter is true, then flow of metabolic waste products might not be reversible by shunting. We hypothesized that hydrocephalus is associated with narrowing of the extracellular space and that chronic hydrocephalus is associated with changes in the extracellular matrix composition. The goals of this experiment were to define the compositional changes of rat brain extracellular matrix constituents in neonatal acute and subacute, juvenile subacute and chronic hydrocephalus rats in comparison to age-matched controls, and to examine anatomical and functional changes in the extracellular compartment. We studied the chondroitin/dermatan sulfate proteoglycans: phosphacan [
18], neurocan (which is a member of the lectican family) [
19], and NG2 [
20], and the two small leucine-rich proteoglycans: decorin and biglycan [
21]. These extracellular proteins have chains of glycosaminoglycans bound to core proteins. With the additional use of less specific histochemical stains, we covered most of the major classes of extracellular substances in the brain. We also studied the basal lamina-associated protein laminin [
22]. We used freeze-substitution electron microscopy [
23,
24] to assess relative dimensions of the cortical extracellular space. This method is one of the only ways to assess the physical characteristics of the extracellular compartment independent of its functional status by avoiding the cellular swelling that occurs using conventional fixation methods [
25,
26]. It is, however, limited in the depths to which preservation is acceptable.