Loss of substance P and inflammation precede delayed neurodegeneration in the substantia nigra after cerebral ischemia
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.
References (35)
- et al.
Risk of stroke in patients with idiopathic Parkinson disease
Parkinsonism Related Disorders
(2010) - et al.
Inflammation in areas of remote changes following focal brain lesion
Progress in Neurobiology
(2005) - et al.
The GABA and substance P input to dopaminergic neurones in the substantia nigra of the rat
Brain Research
(1990) - et al.
Inflammation and brain injury: acute cerebral ischaemia, peripheral and central inflammation
Brain, Behavior, and Immunity
(2010) - et al.
Focal ischemia induces transient expression of IL-6 in the substantia nigra pars reticulata
Brain Research
(2001) - et al.
Synaptic connections between pars compacta and pars reticulata neurones: electrophysiological evidence for functional modules within the substantia nigra
Brain Research
(1994) - et al.
TTC, fluoro-jade B and NeuN staining confirm evolving phases of infarction induced by middle cerebral artery occlusion
Journal of Neuroscience Methods
(2009) - et al.
Tumor necrosis factor-[alpha] expression in areas of remote degeneration following middle cerebral artery occlusion of the rat
Neuroscience
(2003) - et al.
A quantitative morphometrical study of neuron degeneration in the substantia nigra in Parkinson’s disease
Journal of the Neurological Sciences
(1996) - et al.
Decrease of substance P-like immunoreactivity in the substantia nigra and pallidum of parkinsonian brains
Brain Research
(1983)
Inflammation and neurodegeneration in Parkinson’s disease
Parkinsonism Related Disorders
Exo-focal postischemic neuronal death in the rat brain
Brain Research
Inflammation and glial responses in ischemic brain lesions
Progress in Neurobiology
Neuroinflammatory mechanisms in Parkinson’s disease: potential environmental triggers, pathways, and targets for early therapeutic intervention
Experimental Neurology
A substance P antagonist improves outcome when administered 4 h after onset of ischaemic stroke
Brain Research
Gabaergic transmission and tyrosine hydroxylase expression in the nigral dopaminergic neurons: an in vivo study using a reversible ischemia model of rats
Neuroscience
Detection and quantification of remote microglial activation in rodent models of focal ischaemia using the TSPO radioligand CLINDE
European Journal of Nuclear Medicine and Molecular Imaging
Cited by (53)
Progressive secondary exo-focal dopaminergic neurodegeneration occurs in not directly connected midbrain nuclei after pure motor-cortical stroke
2020, Experimental NeurologyCitation Excerpt :This is suggested by reports of early microglia activation and in some studies associated inflammatory markers in the SN compared to the more delayed time course of neuronal cell loss after tMCAO. First microglial recruitment or morphological signs of activation and enhanced TNFalpha expression in the SN were detected between 3 and 7 days after tMCAO in rats (Dihné and Block, 2001; Korematsu et al., 1995; Loos et al., 2003; Uchida et al., 2010) and already after 24 h in mice (Rodriguez-Grande et al., 2013). This consistently preceded the structural end result of neurodegeneration, neuronal (NeuN or Nissl) or dopaminergic (TH-ir) cell loss in the SN.
Mitochondrial and calcium perturbations in rat CNS neurons induce calpain-cleavage of Parkin: Phosphatase inhibition stabilizes pSer <sup>65</sup> Parkin reducing its calpain-cleavage
2019, Biochimica et Biophysica Acta - Molecular Basis of DiseaseIs hyperhomocysteinemia associated with the structural changes of the substantia nigra in Parkinson's disease? A two-year follow-up study
2019, Parkinsonism and Related DisordersCitation Excerpt :Although PD is recognized as a neurodegenerative disease, there is increasing evidence that vascular factors play an important role in its development [1]. As early as 2013, animal studies have shown that ischemic lesions of the striatum may affect the substantia nigra (SN) and accelerate the progress of PD [2]. Moreover, our previous study found that striatal silent lacunar infarction (SSLI) was more likely to occur in PD patients with hyperhomocysteinemia (HHCY).