T. 11 3846.3272 / 3846.3273 | contato@cukiert.com.br
Rua Dr. Alceu de Campos Rodrigues, 247 - 12° and. Cj. 21
SĂŁo Paulo/SP - Brasil - CEP 04544-000

ClĂ­nica de Epilepsia | Trabalhos na ĂŤntegra

Extracellular matrix components are altered in the hippocampus, cortex, and cerebrospinal fluid of patients with mesial temporal lobe epilepsy.


Summary:  Purpose: This work studied the profile of glycosaminoglycans (GAGs) in the hippocampus, cortex, and cerebrospinal fluid of patients with temporal lobe epilepsy (TLE).

Methods: The GAGs were analyzed by agarose gel electrophoresis, enzymatic degradation, and enzyme-linked immunosorbent assay (ELISA).

Results: The hippocampus of TLE patients showed increased levels of chondroitin sulfate and hyaluronic acid against normal levels of these GAGs in the neocortex and cerebrospinal fluid (CSF).

Conclusions: These results suggest that these matrix components could be involved in the pathophysiology of TLE.

The glycosaminoglycans (GAGs) are found in extracellular matrix of all tissues and, except for hyaluronic acid (HA), they exist as proteoglycans. These compounds can also be found as membrane receptors bound to extracellular matrix proteins. The versatility of GAGs and their ability for multiple interactions with other molecules enables them to function as a “glue” in cellular interaction, capturing soluble molecules such as growth factors into the matrix and cell surface. HA has been related to aggregation, proliferation, migration, and adhesion of cells.

The most frequent pathologic finding in temporal lobe epilepsy (TLE) is mesial sclerosis with intense neuronal loss and gliosis in several hippocampal subfields. Mossy fiber sprouting into the inner molecular layer of dentate gyrus, reactive synaptogenesis, and axonal circuit reorganization can also be found in the hippocampus of mesial TLE patients.

Accordingly, we present the profile of chondroitin sulfate (CS), heparan sulfate (HS), and HA levels in the cortex and hippocampus and cerebrospinal fluid (CSF) of patients with refractory mesial TLE.


The surgery was performed in TLE patients as previously reported, and the neocortices (n = 8) and hippocampi (n = 8) were stored at –70°C for biochemical assay. The GAG concentration was compared with those obtained from similar brain regions, removed during autopsy of patients with no evidence of pathology on the basis of gross and routine histologic examination. CSF samples of patients with TLE (n = 8) were obtained 24 h before surgery by lumbar spinal tap and immediately frozen to quantify HA. The CSF used as control was negative for neurocysticercosis, obtained from patients with a clinical tensional headache with normal neurologic and computerized tomography (CT) examination (n = 8). The GAGs were identified and quantified by a combination of agarose gel electrophoresis and enzymatic degradation, as previously described. HA from cerebral cortex, hippocampus, and CSF was measured by a fluoroassay using HA-binding proteins isolated from bovine cartilage as immobilized on microwell plate (solid phase) as biotinylated probe. Student's t test was used to study the CS, HS, and HA concentration from hippocampus, neocortex, and CSF, and p < 0.05 was accepted as significant.


The quantification of hippocampal GAG levels obtained from epileptic tissues showed an increased amount of 224% of CS (20.40 ± 9.61 μg CS/mg dry tissue; p = 0.0109) when compared with control values (9.13 ± 2.39 μg CS/mg dry tissue). The epileptic tissue also showed an increased concentration of HA (2,386.8 ± 561.4 μg HA/mg dry tissue; 146%; p = 0.039) when compared with control values (1,638.8 ± 385.3 μg HA/mg dry tissue). A similar concentration of HS was found in the hippocampus of TLE patients (4.6 ± 2.04 μg HS/mg dry tissue), when compared with control (5.40 ± 2.66 μg HS/mg dry tissue; p = 0.51). Furthermore, no alterations were found in CS (11.15 ± 9.14 μg CS/mg dry tissue; p = 0.65), HS (2.41 ± 0.77 μg HS/mg dry tissue; p = 0.72), and HA (1,517.4 ± 411.34 μg AH/mg dry tissue; p = 0.95) concentration in the cortex of TLE patients, when compared with control (9.49 ± 5.70 μg CS/mg dry tissue; 2.26 ± 0.91 μg HS/mg dry tissue; 1,542.2 ± 689.73 μg HA/mg dry tissue, respectively). The quantification of HA in the CSF of patients with TLE showed values (205.63 ± 175.06 ng/ml CSF) similar to those of the control group (198.78 ± 99.07 ng/ml CSF (p = 0.95; Fig. 1).

Figure 1. Profile of glycosaminoglycans (GAGs) in the cortex, hippocampus, and cerebrospinal fluid of temporal lobe epilepsy (TLE) patients. The glycosaminoglycans were quantified as previously described. CS, chondroitin sulfate; HS, heparan sulfate; HA, hyaluronic acid; C, control tissue; CTX, cortex; HPC, hippocampus; CSF, cerebrospinal fluid. **p = 0.0109; *p = 0.0395.


We showed, for the first time, changes in GAGs concentration in the brain of TLE patients. A selective increase of CS and HA was found in the hippocampus associated with normal levels of these GAGs in the neocortex. Similar results were found by Naffah-Mazzacoratti et al, who reported changes in GAG levels in the hippocampus of rats subjected to pilocarpine-induced epilepsy.

CS is one of the more important matrix components in the nervous system during the postnatal period. Although proteoglycans rich in CS (CSPG) have been shown to regulate or guide neurite outgrowth in the developing brain, their role in neonatal and in adult brain is uncertain. CS or CSPG has been related to a reduction in delayed neuronal death, induced by excitatory amino acid exposure. The increased CS level found in the hippocampus of TLE patients could be a glial response to the increased release of glutamate during seizures.

Conversely, the biologic effects of HS have been associated with fibroblast growth factors (FGFs), which belong to a family of heparin-binding proteins. A hypothesis is that the HS acting as FGF co-receptor regulates the binding of this compound to its receptor, modulating its function. If FGF function really depends on HS concentration, the normal levels of this GAG found in the cortex and hippocampus of TLE patients suggests that FGF action is preserved in these tissues.

The brain contains extracellular matrix rich in HA as well as a number of hyaluronan-binding proteins such as versican, brain-enriched hyaluronan-binding protein, hyaluronection, and CD44. According to Koochekpour, the HA/CD44H interaction induces cell detachment and stimulates migration and invasion of glioma cells in vitro. Our work shows increased concentration of HA in the hippocampus. This result suggests that HA could be produced by reactive gliosis, and this overproduction may be associated with cell detachment, necessary for axon migration during mossy fiber sprouting. The normal level of HA in neocortex is reflected by normal levels of this GAG in CSF.


The increased concentration of HA as well as CS in the hippocampus of TLE patients shows the importance the matrix compounds during neosynaptogenesis, neurite outgrowth, and the mossy fiber sprouting found in TLE phenomena.

Acknowledgment: This study was supported by FAPESP, PRONEX, CNPq, and CAPES.



Barnea G, Grumet M, Milev P, et al. Receptor tyrosine phosphatase β is expressed in the form of proteoglycan and binds to the extracellular matrix tenascin. J Biol Chem 1994;20:14349–52.

Ruoslahti E, Yamaguchi Y. Proteoglycan as modulators of growth factors activities. Cell 1991;64:867–9.

Fraser JRE, Laurent TC, Laurent UBG. Hyaluronan: its nature, distribution, functions and turnover. J Intern Med 1997;242:27–33.
Direct Link:

Mathern GW, Babb TL, Leite JP, et al. The pathogenic and progressive features of chronic human hippocampal epilepsy. Epilepsy Res 1996;26:151–61.

Naffah-Mazzacoratti MG, Amado D, Cukiert A, et al. Monoamines and their metabolites in cerebrospinal fluid and temporal cortex of patients with epilepsy. Epilepsy Res 1996;25:133–7.

Nader HB, Dietrich CP, Buonassisi V, et al. Heparin sequences in the heparan sulfate chains of an endothelial cell proteoglycan.Proc Natl Acad Sci U S A 1987;84:3565–69.

Nader HB, Porcionato MA, Tersariol ILS, et al. Purification and substrate specificity of heparitinase I and heparitinase II fromFlavobacterium heparinum: analyses of heparin and heparan sulfate degradation products by 13C NMR spectroscopy. J Biol Chem 1990;265:16807–13.

Dietrich CP, Silva ME, Michelacci YM. Sequential degradation of heparin in Flavobacterium heparinum: purification and properties of five enzymes involved in heparin degradation. J Biol Chem 1973;248:6408–15.

Dietrich CP, Dietrich SM. Electrophoresis behaviour of acidic mucopolysaccharides in diamine buffers. Anal Biochem1976;70:645–7.

Naffah-Mazzacoratti MG, Argañaraz GA, Porcionatto MA, et al. Selective alteration of glycosaminoglycans synthesis and proteoglycan expression in rat cortex and hippocampus in pilocarpine-induced epilepsy. Brain Res Bull 1999;50:22–39.

Jenkins HG, Bachelard HS. Developmental and age-related changes in rat brain glycosaminoglycans. J Neurochem1988;51:1634–40.
Direct Link:

Chisamore B, Solc M, Dow K. Excitatory amino acid regulation of astrocyte proteoglycan. Dev Brain Res 1996;97:22–8.

Okamoto M, Mori S, Endo H. A protective action of chondroitin sulfate proteoglycans against neuronal cell death induced by glutamate. Brain Res 1994;637:57–67.

Meldrum B, Garthwaite J. Excitatory amino acid neurotoxicity and neurodegenerative diseases. TIPS 1990;11:379–87.

Small DH, Mok SS, Williamson TG, et al. Role of proteoglycans in neuronal development, regeneration and aging brain. J Neurochem 1996;67:889–99.
Direct Link:

Yayon A, Klagsbrum M, Esko JD, et al. Cell surface heparin-like molecules are required for basic fibroblast growth factor to its high affinity receptor. Cell 1991;64:841–8.

Nagy JI, Prince ML, Stainess WA, et al. The hyaluronic acid receptor RHAMM in noradrenergic fibers contributes to axon growth capacity of locus ceruleus neurons in an intraocular transplant model. Neuroscience 1998;86:241–55.

Koochekpor S, Pilkington GJ, Merzak A. Hyaluronic acid/CD44H vitro interaction induces detachment and stimulates migration and invasion of human glioma cells. Int J Cancer 1995;63:450–4.
Direct Link: