Induction of apoptosis by cerebrospinal fluid from patients with primary-progressive multiple sclerosis in cultured neurons Alberto Alca ´zar a , Ignacio Regidor b , Jaime Masjuan b , Matilde Salinas a , Jose ´ C. A ´ lvarez-Cermen ˜o b,c, * a Departamento de Investigacio ´ n, Hospital Ramo ´ n y Cajal, Madrid, Spain b Servicio de Neurologı´a, Hospital Ramo ´ n y Cajal, Madrid, Spain c Department of Medicine, Universidad de Alcala ´ , Alcala ´ de Henares, Spain Received 12 June 1998; received in revised form 25 August 1998; accepted 28 August 1998 Abstract We have studied the noxious effect of cerebrospinal fluids (CSF) from patients with primary-progressive multiple sclerosis (MS) on cultured neurons. Cells were exposed to CSF for 8 days and the possible neuronal damage was determined. Morpho- logical studies with phase-contrast microscopy showed cellular shrinkage indicating apoptosis. CSF-induced apoptosis as evidenced by the fluorescent DNA-binding dye Hoechst 33342, as well as by the TUNEL-reaction, was only present in pri- mary-progressive MS patients with a worsening disease. This neuron injury did not correlate with blood–brain barrier dysfunction nor with intrathecal IgG synthesis. On the contrary, CSF from either stable primary-progressive or other non-inflammatory neurological diseases, did not induce any culture damage. Undetectable or low similar tumor necrosis factor-alpha (TNF-a) levels (range to 8.7 pg/ml) were found in the CSFs tested regardless they damage cultures or not. These results suggest that soluble factors, other than TNF-a, molecules transudated from blood or IgG, present in the CSF of active primary-progressive patients with MS induce neuronal apoptosis.  1998 Elsevier Science Ireland Ltd. All rights reserved Keywords: Multiple sclerosis; Primary-progressive multiple sclerosis; Apoptosis; Cultured neurons Despite extensive research in recent years, the precise mechanism causing tissue damage in multiple sclerosis (MS) remains elusive. The study of MS lesions has revealed T-cell infiltration and upregulation of several cytokines [3] which, at least in vitro, have been shown to induce myelin injury and oligodendrocyte apoptosis [13,17]. Further macrophage and resident microglia activation [7] and local production of reactive oxygen intermediates are part of an inflammatory cascade which produces myelin break- down, local edema, and homing of new infiltrating lympho- cytes to the lesion [4]. Nevertheless, magnetic resonance imaging studies indicate that severe demyelination can occur in MS patients who are asymptomatic [6]. It seems likely that in addition to a hypothetical specific attack against myelin or the oligodendrocyte, other neighboring cells, such as neurons, could be damaged by soluble med- iators of inflammation in a non-specific fashion [15]. Since such events in MS are often reflected by the presence of inflammatory mediators in the cerebrospinal fluid (CSF) [14], we considered it of interest to investigate whether the CSF from patients with MS would damage cortical neu- rons in primary cultures. We report for the first time that CSF from active patients with primary-progressive MS induces neuronal apoptosis in cultured neuronal cells. The effect of eleven different CSF samples on cultured neurons was studied. Five of these samples were obtained from patients with clinically definite primary-progressive (PP) MS, whilst the remaining six were from patients with other non-inflammatory neurological diseases (OND). The clinical diagnosis of each of these OND patients included ischemic III and VI nerve palsies, bulbar paralysis, cervical compressive myelopathy and two thrombotic strokes. All samples were obtained by lumbar puncture (LP), performed Neuroscience Letters 255 (1998) 75–78 0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(98)00708-3 * Corresponding author. Fax: +34 91 3369016; e-mail: jose.c.alvarez@hrc.es