Neuroscience Letters 260 (1999) 17–20
Propentofylline attenuates microglial reaction in the rat spinal cord induced by middle cerebral artery occlusion Yun-Ping Wu a, Amanda McRae b, Karl Rudolphi c, Eng-Ang Ling a ,* a
Department of Anatomy, Faculty of Medicine, National University of Singapore, Singapore 119260, Singapore b Department of Preclinical Science, University of the West Indies, St. Augustine, Trinidad and Tobago c Hoechst AG, Frankfurt am Main, Germany Received 15 October 1998; received in revised form 13 November 1998; accepted 13 November 1998
Abstract This study examines the effect of Propentofylline (PPF) on reactive microglia in the lumbar spinal cord in rats following focal cerebral ischaemia produced by permanent occlusion of the middle cerebral artery (MCA). Our results showed that daily treatment of PPF beginning at 24 h after MCA occlusion for 2 or 4 consecutive days markedly suppressed the microglial response as detected immunohistochemically with OX-42. The most dramatic effect was the prevention of transformation of ramified microglia into amoeboidic form as well as formation of perineuronal microglia in close association with the soma of motoneurons. This has greatly amplified the potentiality of PPF used as a neuroprotective drug against microglia-related neuron damage induced by cerebral ischaemia. 1999 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Propentofylline; Adenosine; Immunohistochemistry; Microglial activation; Focal cerebral ischaemia; Lumbar spinal cord
Despite intense research aimed at the development of effective therapeutic interventions, the means of preventing ischaemic brain damage are as elusive as ever. Among numerous therapies under current testing, approaches based on the balance between A1 and A2 receptor-mediated adenosine action offer a substantial promise in reducing ischaemia-related structural and functional damage . A late decline in neurologic function that occurs days after an ischaemic insult to the central nervous system is often associated with progressive damage to neurons . Although the mechanisms to account for such delayed deterioration in function remain uncertain, de novo production of cytotoxic substances may be involved . One widely held view is that cytotoxic molecules such as excitatory amino acids and short-lived free radicals [2,5] which participate in contact-mediated killing of neurons  are not only released by damaged neuronal tissues, but also by reactive microglia. * Corresponding author. Fax: +65 778764; e-mail: [email protected]
0304-3940/99/$ - see front matter PII S0304- 3940(98) 00941- 0
The xanthine derivative Propentofylline (PPF) (1-(5′oxohexyl)-3-methyl-7-propylxanthine) protects against delayed post-ischaemic hippocampal damage following global forebrain ischaemia in gerbils , and reduces infarct volume and neurological symptoms when given 15 min after permanent middle cerebral artery (MCA) occlusion in rats . It favours adenosine A2 receptor actions by inhibiting the adenosine membrane transporter  and by blocking phosphodiesterases. . While it is unequivocal that PPF protects against microglia-related brain damage in the hippocampus after brain ischaemia [4,8], it remains uncertain if the drug would also affect the microglial reaction far removed from the primary site of infarct such as that in the lumbar spinal cord induced by MCA occlusion . This is deemed to be a critical issue since some of the spinal neurons to which the reactive microglia associated with were motoneurons innervating the limb musculature . This study was therefore aimed to find out whether in vivo treatment with the drug could interfere with an ongoing pathological activation of microglial cells. Experiments were carried out on a total of 20 male Wistar
1999 Elsevier Science Ireland Ltd. All rights reserved
Y.-P. Wu et al. / Neuroscience Letters 260 (1999) 17–20
Fig. 1. (A) OX-42 immunostained reactive microglia in the ipsilateral frontoparietal cortex show hypertrophied (arrowheads) or rod-like (arrows; see also inset) cell bodies, 3 days after MCA occlusion. In the corresponding areas in PPF-treated rats (B), the cells are almost totally depleted. The majority of the microglia at the site of ischaemia display OX-42 immunoreactivity comparable to normal levels (see peripheral parts of section). Scale bar, 300 mm.
rats weighing between 280 and 350g body weight. Following anaesthesia with an intraperitoneal injection of chloral hydrate (300 mg/kg), the right MCA was permanently occluded as described previously . In rats receiving PPF injection, treatment began at 24 h after MCA occlusion; each rat was given daily i.p. injections of 10 mg/kg PPF and continued for 2 or 4 consecutive days. Control rats received equivalent injections of the vehicle (phosphate buffered saline, pH 7.6). The rats in both groups were sacrificed at 3 (n = 5) and 5 (n = 5) days after MCA occlusion. They were perfused transcardially first with 0.1 M phosphate buffer (PB, pH 7.4) followed by 4% paraformaldehyde. Serial 40 mm-thick coronal sections of the brain and transverse sections of the lumbar spinal cord were cut on a cryostat. Alter-
nate sections were collected and incubated in the monoclonal antibody OX-42 (Sera Lab, MAS370b) which recognizes the complement type 3 receptor (CR3) on microglia. The sections were processed for immunocytochemistry using the avidin-biotin-peroxidase complex method (Vector). In agreement with others , besides cortical neurons undergoing ischaemic changes such as pyknosis and microvacuolation, many reactive microglia, as identified by their enlarged cell body and intense OX-42 immunoreactivity, were detected in the ipsilateral frontoparietal cortex at 3 days after MCA occlusion. Some of the reactive microglia were extremely elongated with their rod-like cell bodies arranged normal to the cerebral surface (Fig. 1A). Follow-
Fig. 2. Lumbar enlargement of spinal cord, 3 days after MCA occlusion (A,B) and after PPF treatment (C,D). The OX-42 immunostaining in both the dorsal (A,B) and ventral (A) horns contralateral (right side of pictures) to MCA occlusion is markedly enhanced. Note many ramified microglia display enhanced OX-42 immunoreactivity when compared with cells in the ipsilateral side. Furthermore, scores of amoeboidic microglia (arrows) appear to be distributed preferentially in the medial area of the dorsal horn (B). Following PPF treatment, the reactive microglia notably the amoeboidic form in the corresponding area have diminished (C,D). Scale bars, (A,C) 100 mm; (B,D) 50 mm.
Y.-P. Wu et al. / Neuroscience Letters 260 (1999) 17–20
Fig. 3. Lumbar enlargement of spinal cord, 5 days after MCA occlusion (A,B) and after PPF treatment (C,D). The reactive OX-42 positive microglia in the ventral horn appear to encircle the soma of motoneurons (arrows) (B); such configuration, however, is not evident in the corresponding area after PPF treatment (C,D). The cells instead appear to be evenly distributed. Scale bar, (A,C) 100 mm; (B,D) 50 mm.
ing PPF treatment, microglial activation was noticeably depressed as shown by the almost complete diminution of hypertrophic and rod-like OX-4 immunoreactive cells even in rats receiving two injections (Fig. 1B). As reported previously by us , MCA occlusion induced vigorous microglial response in the lumbar spinal grey matter contralateral to the arterial occlusion (Figs. 2 and 3). The reactive microglia were distributed mainly in Rexed’s laminae I, II and III of the dorsal horn and lamina IX of the ventral horn at 3 days after MCA occlusion (Fig. 2A) whence some of them assumed an amoebodic form (Fig. 2AB). At 5 days the reactive microglia were most conspicuous in the medial area of laminae I, II and III of the dorsal horn and lamina IX of the ventral horn (Fig. 3A). In the latter, the reactive microglia appeared to encircle the soma of motoneurons (Fig. 3B). Daily treatment with PPF markedly depressed the reactive microglia in rats killed at 3 (Fig. 2C,D) and 5 (Fig. 3C,D) days. A remarkable alteration was the diminution of amoebodic microglia at 3 days (Fig. 2D). Furthermore, the reactive microglia appeared to dissociate from the soma of motoneurons so that the cells now with reduced OX-42 immunoreactivity were evenly distributed (Fig. 3D). In agreement with previous study , drastic microglial reaction occurred not only in the primary site of ischaemia in the cerebral cortex, but was also induced in the lumbar spinal cord after MCA occlusion. As the latter is far removed from the primary site of arterial occlusion, it stands to reason that the observed changes must be attributed to the ischaemic insult affecting directly or indirectly the corticospinal neurons. This is because many of the lumbar spinal cord neurons are postsynaptic to the corticospinal neurons
via the corticospinal tracts  which are important pathways with glutamatergic neurotransmission . In the early stages of MCA occlusion, neurons at the primary site of ischaemia have been described to release excitatory amino acids such as glutamate, and activation of N-methyl-daspartate (NMDA) receptors . The NMDA receptors are linked to calcium channels, glutamate and influx of Ca2+  all of which may result in excitotoxicity leading to neuronal degeneration and glial cell activation . In this context, it is suggested that the cortical neuronal death immediately after MCA occlusion may have resulted from ischaemic damage while the delayed neuronal damage in the spinal cord may be attributed to transneuronal degeneration as reported recently by us . In both instances the microglial reaction was effectively suppressed by PPF. It remains to be determined if there is any distinction in the mechanism of PPF action on microglial reaction in neuroprotective action in both areas. It can only be postulated that PPF, possibly by favouring adenosine A2 receptor actions, can interfere with or modify the reactive microglia and change their properties in both immediate and delayed neuronal damage. The functional significance of microglial response in the lumbar spinal cord following MCA occlusion remains unclear. Our  immunoelectron microscopic study has shown that some of the reactive microglia particularly those in the dorsal horn were engaged in the phagocytosis of dying neurons. It has also been reported that activated microglia in vitro are capable of releasing several potentially cytotoxic substances, e.g. free radicals, excitatory amino acids, etc. It seems justified to suggest that the reactive microglia here described would also release similar
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substances which may be harmful to the spinal neurons. Thus, the effective suppression of ischaemia induced microglial response by PPF in the lumbar spinal cord indicates its potency and efficacy as a neuroprotective drug. Results from in vitro studies have shown that PPF not only depresses the high-level production of reactive oxygen intermediates in microglia-derived macrophages , but also prevents their transformation into macrophages and inhibits proliferation . The lack of amoeboidic reactive microglia following PPF treatment is therefore relevant in this context. It is possible that the drug has suppressed the transformation of microglia from the ramified to amoeboidic form as active macrophages. PPF has been reported to inhibit the presynaptic release of the potentially neurotoxic transmitter glutamate , as well as enhancing the synthesis and release of nerve growth factor (NGF) by cultured mouse astroglial cells . It is speculated that these may also contribute to the neuroprotective actions for the cortical and spinal cord neurons in the present experimental model. The present study has demonstrated that prolonged treatment with PPF can interfere with the pathological activation of microglial cells. Thus, it may provide a neuroprotective therapy for those diseases in which an ongoing activation of microglial cells is thought to be relevant for neuronal damage. This work is supported by a research grant (RP950363) from the National University of Singapore.  Banati, R.B., Schubert, P., Rothe, G., Gehrmann, J., Rudolphi, K., Valet, G. and Kreutzberg, G.W., Modulation of intracellular formation of reactive oxygen intermediates in peritoneal macrophages and microglia/brain macrophages by propentofylline, J. Cereb. Blood Flow Cell Metab., 14 (1949) 145–149.  Colton, C.A. and Gilbert, D.L., Production of superoxide by a CNS macrophage, the microglia, FEBS Lett., 223 (1987) 284– 288.  Collaco, M.Y., Aspey, B.S., Belleroche, J.S. and Harrison, M.J., Focal ischaemia causes an extensive induction of immediate early genes that are sensitive to MK-801, Stroke, 25 (1994) 1855–1860.  Deleo, J., Toth, L., Schubert, P., Rudolphi, K. and Kreutzberg, G.W., Ischaemia induced neuronal cells death, calcium accumulation, and glial response in the hippocampus of the mongolian gerbil and protection by propentofylline (HWA 285), J. Cereb. Blood Flow Metab., 7 (1987) 745–752.  Giulian, D. and Baker, T.J., Characterization of amoeboid microglia isolated from developing mammalian brain, J. Neurosci., 6 (1986) 2163–2178.
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