Loss of substance P and inflammation precede delayed neurodegeneration in the substantia nigra after cerebral ischemia

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Abstract

Focal cerebral ischemia leads to delayed neurodegeneration in remote brain regions. The substantia nigra (SN) does not normally show primary neuronal death after ischemic events affecting the striatum, but can exhibit delayed neuronal loss after the ischemic injury through mechanisms that are unknown. No data are available in mice showing acute post-stroke inflammation and remote injury in the SN. Substance P (SP), a mediator of neurogenic inflammation, is a key element of the striato-nigral circuitry, but alterations of SP in the SN have not been studied after acute striatal injury. Inflammation, a key contributor to neuronal death, is found in the SN after striatal ischemia, but it is unknown whether it precedes or occurs concomitantly with neuronal death. We hypothesised that focal striatal ischemia induces changes in SP levels in the SN and that inflammation precedes neuronal death in the SN. Using the middle cerebral artery occlusion model, we found a significant loss of SP in the ipsilateral SN 24 h after striatal ischemia in mice. In the same area where SP loss occurs, significant glial and vascular activation, but no neuronal death, were observed. In contrast, a marked neuronal loss was observed within six days in the area of SP loss and inflammation. Our data suggest that focal loss of SP and early inflammatory changes in the SN precede remote neuronal injury after striatal ischemic damage. These observations may have important implications for motor impairment in stroke patients and indicate that striatal ischemia might facilitate Parkinson’s disease development.

Highlight

► Early loss of substance P in the substantia nigra (SN) is accompanied with inflammation after experimental stroke and precedes cell death in the SN.

Introduction

Cerebral ischemia that occurs in stroke induces rapid neuronal death in the core of the infarct, followed by secondary neuronal injury triggered by inflammation that evolves in the surrounding hypoperfused region. In addition to this neuronal injury, cerebral ischemia also triggers changes in remote areas which are connected to the ischemic core. Indeed, clinical and experimental data from rat models indicate that neuronal death and inflammation also occur in remote areas such as the substantia nigra (SN) (Block et al., 2005). The SN is a key structure involved in motor control, and is strongly affected in Parkinson’s disease (PD). The SN has bi-directional neuronal connections with the striatum, which becomes ischemic after occlusion of the middle cerebral artery (MCA). Striatal neurons innervate the SN pars reticulata (SNr), where they release both substance P (SP) and GABA (Kopell et al., 2006). The SNr partially innervates the SN pars compacta (SNc) (Hajos and Greenfield, 1994), and the SNc projects dopaminergic neurons to the dorsal striatum, regulating voluntary motor activity (Kopell et al., 2006, Saklayen et al., 2004). Stroke patients with striatal, but not cortical, damage display SN degeneration (Nakane et al., 1992, Ogawa et al., 1997) and middle cerebral artery occlusion (MCAo) in rat induces extensive delayed neurodegeneration and inflammation in the SN from 1 to 20 weeks after the onset of ischemia (Dihné and Block, 2001, Loos et al., 2003, Nagasawa and Kogure, 1990, Uchida et al., 2010).

Although the mechanisms underlying remote injury are largely unclear, it has been suggested that changes in neurotransmitter signalling can contribute to remote injury. Indeed, post-ischemic alterations of the striato-nigral GABAergic system are associated with neuronal death in the SN and later motor deficits (Lin et al., 2010). Another key striato-nigral neurotransmitter is SP. SP belongs to the tachykinin family and binds to the neurokinin-1 (NK1) tachykinin receptor (NK1R), both of which are expressed at highest levels in the SN (Bolam and Smith, 1990, Whitty et al., 1997) and play a crucial role in the modulation of motor functions. In addition, SP is a well known mediator of neurogenic inflammation (O’Connor et al., 2004), a process through which released neuropeptides increase vascular permeability, facilitating the development of an inflammatory response. SP alterations have been observed in the core of the damage in the brain (Turner et al., 2006). However, no reports have studied remote alterations of SP after striatal ischemia and its effects in remote inflammation and neuronal injury.

Neuronal cell death in the ischemic striatum is profoundly influenced by inflammation (Hossmann, 2006, Jin et al., 2010), which is an important therapeutic target after stroke (Denes et al., 2010). Activation of glial cells (Stoll et al., 1998), upregulation of vascular adhesion molecules and disruption of the blood–brain barrier, which allow circulating leukocytes to infiltrate the cerebral tissue (Ley et al., 2007), are hallmarks of the inflammatory response, some of which are observed in the SN weeks after MCAo (Block et al., 2005, Uchida et al., 2010). However, whether early inflammatory changes precede and contribute to neuronal death in the SN after MCAo is completely unknown.

Therefore, we hypothesised that cerebral ischemia affecting the striatum might alter SP-ergic innervation or SP levels in the SN, and induces early inflammatory changes that may contribute to delayed neuronal death. Our results show that focal loss of SP and inflammation occur in anatomically corresponding areas of the SN early after MCAo preceding significant neuronal death.

Section snippets

Animals

This study used 10–18 week-old male C57/BL6 mice, weighing 25–30 g. All animal procedures were performed under Home Office license (UK) and adhered to regulations specified in the Animals (Scientific Procedures) Act (1986). Mice were kept at 21 °C ± 1 °C and 65% humidity) with a 12 h light–dark cycle with free access to food and water.

Middle cerebral artery occlusion and motor assessment

Mice were anaesthetised by inhalation of 4% isofluorane, and anaesthesia was maintained with 1.5% isofluorane during surgery. Core body temperature was maintained at 37.0

Focal ischemic damage results in profound morphological alterations of NK1R-positive nerves in the striatum

Within the healthy striatum, we detected NK1R immunoreactive (ir) neurons co-localised with SP-positive nerve terminals (Fig. 1A). 45 min MCAo induced marked morphological changes in the NK1R-ir neuronal processes within 24 h in the ipsilateral striatum including swelling of boutons, and an increase in NK1R expression (Fig. 1B). Cresyl violet staining revealed a marked reduction of viable neurons in the ipsilateral striatum and cortex (Fig. 1B), confirming the presence of focal ischemic damage.

Discussion

In order to better understand mechanisms of remote injury in the SN following transient focal ischemia, we investigated whether changes in SP levels and inflammation occur in the SN preceding neuronal injury. To date, no studies investigated early changes in remote neuronal injury and inflammation in the SN after focal cerebral ischemia in mice, and no studies addressed local variations of SP levels in the SN after stroke. We report here for the first time a loss of SP in the SN after transient

Acknowledgments

We are grateful for funding provided by the European Union’s Seventh Framework Programme (FP7/2008–2013) under Grant agreements Nos. 201024 and 202213 (European Stroke Network) and the Simon and Simone Collins studentship. We would like to thank Professor Nancy Rothwell for critically reviewing the manuscript.

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