Blocking of leukocyte accumulation in the cerebrospinal fluid augments bacteremia and increases lethality in experimental pneumococcal meningitis

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Abstract

The role of leukocyte accumulation in the cerebrospinal fluid (CSF) in the evolution of the pathophysiological changes that occur in bacterial meningitis is unclear. Here, we investigate how leukocyte recruitment to the CSF, modulated by the leukocyte blocker fucoidin, affects the extent of brain damage and outcome in pneumococcal meningitis in rats treated with ceftriaxone from 28 h after infection.

Rats treated with fucoidin from time of infection had an excess risk of a fatal outcome compared to rats not receiving fucoidin (25 / 63 versus 5 / 34, p = 0.012), whereas the risk of cortical damage in surviving animals was comparable (16 / 44 versus 9 / 29, p = 0.8). Pre-treatment with fucoidin attenuated CSF pleocytosis 24 h after infection (median 400 versus 800 × 106 cells/l, p = 0.01) without affecting CSF bacterial counts (2.3 × 105 versus 3.6 × 105 CFU/ml, p = 0.54). A significant increase in blood bacterial counts was found among rats pre-treated with fucoidin (median 9.6 × 102 versus 5.2 × 102 CFU/ml, p = 0.03). Furthermore, blood bacterial count was found to be an important predictor of fatal outcome as shown by multivariate logistical regression analysis (OR 4.43, 95% CI [1.16–17.0] p = 0.03).

In summary, blocking leukocyte entry to the central nervous system in experimental pneumococcal meningitis compromises the survival prognosis but does not affect the risk of brain damage or level of infection in this compartment. Conversely, poorer prognosis was associated with an increase in bacterial load in blood, suggesting that leukocyte blockage affects the host's ability to control systemic infection.

Introduction

Bacterial meningitis is characterized by an inflammatory response, altered blood brain barrier function and brain damage caused by an invading pathogen within the subarachnoidal space. Whilst the exact mechanisms responsible for these changes are not fully understood, leukocyte infiltration observed in damaged brain tissue, obtained from autopsies of patients with bacterial meningitis or from experimental studies, suggests that leukocytes are involved in the pathogenesis of vasculitis, thrombophlebitis and brain damage (Cairns and Russel, 1946, Quaade and Kristensen, 1962, Pfister et al., 1992, Tuomanen et al., 1989, Zysk et al., 1996). Therefore, attenuation of the inflammatory response has been a primary target in attempts to improve the outcome of bacterial meningitis. Recently, the demonstration of a survival advantage with the use of dexamethasone in a clinical study of bacterial meningitis has indicated the beneficial effect of an anti-inflammatory approach (de Gans and van de Beek, 2002). Indeed the blocking of leukocyte entry into the brain has previously been shown to reduce indices of blood–brain-barrier breakdown (Saez-Llorens et al., 1991, Ostergaard et al., 2000, Tuomanen et al., 1989), and to reduce formation of brain oedema in experimental meningitis models (Tuomanen et al., 1989, Saez-Llorens et al., 1991, Angstwurm et al., 1995, Weber et al., 1995, Weber et al., 1997). In experimental models of stroke leukocyte blocking has been shown to attenuate development of brain damage due to reperfusion injury (Prestigiacomo et al., 1999). However, the extrapolation of these results to a potential effect on mortality, outcome and development of brain damage still remain to be clarified.

The primary aim of this study was therefore to investigate the influence of a reduced leukocyte response in the central nervous system on outcome and brain pathology in an experimental model of pneumococcal meningitis in rats. The selectin blocker fucoidin was used to block leukocyte entry into the cerebrospinal fluid (CSF) since it has previously been shown to efficiently inhibit leukocyte accumulation in the CSF in experimental meningitis in rabbits (Granert et al., 1994, Granert et al., 1998, Granert et al., 1999, Ostergaard et al., 2000).

Section snippets

Experimental pneumococcal meningitis

The experimental protocol was approved by the Danish Animal Inspectorate (Dyreforsoegstilsynet). Young adult male Wistar rats (180–200 g) were used for the meningitis experiments. Normal day/night cycles and free access to food and water were provided.

Experimental study design

A total of 125 rats were assigned to the trials. A total of 108 rats were inoculated with a bacterial suspension and randomly assigned to receive fucoidin 3 times daily from the time of infection (fucoidin pre-treated group, n = 70) or merely

Effect of pre-treatment with fucoidin on WBC and bacterial counts in CSF and blood 24 h after bacterial inoculation (Table 1)

WBC counts in CSF were significantly reduced in fucoidin pre-treated rats compared to controls (p = 0.01) whereas no difference in the WBC counts in blood was observed between the two groups (p = 0.55) (Table 1).

Bacterial counts in CSF were unaffected by pre-treatment with fucoidin (p = 0.54). A significant increase in blood bacterial counts was found among fucoidin pre-treated rats compared to controls (p = 0.03).

Effect of pre-treatment with fucoidin on fatal outcome and sequelae (Table 1 and Fig. 1)

The risk of developing terminal illness was increased in the group pre-treated with

Discussion

Our results demonstrate that the blocking of leukocyte influx into the cerebral compartment increased the risk of a fatal outcome from pneumococcal meningitis, suggesting that migration of leucocytes from the blood compartment is an important component of the host response to bacteria in the CSF. This is consistent with data acquired from other models of infection (e.g. arthritis, sepsis, meningitis) where increased mortality is found in animals with a genetically or medically attenuated

Acknowledgements

Financial support from the following foundations is gratefully acknowledged: Den Lægevidenskabelige Forskningsfond for Storkøbenhavn, Færøerne og Grønland; Eivind Eckbos dansk-norske Legat; Lily Benthine Lunds Fond; Christian Larsen og Dommer Ellen Larsens Legat; Direktør Jacob Madsen og Hustru Olga Madsens Fond; Fonden til Lægevidenskabens Fremme and H:S Fondet. The authors would also like to thank Jesper Madsen for statistical support, Dr. Ian Rowland for help in preparing the manuscript and

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