Abstract
BackgroundSpinal reactive astrocytes and microglia are known to participate to the initiation and maintenance of neuropathic pain. However, whether reactive astrocytes and microglia in thalamic nuclei that process sensory-discriminative aspects of pain play a role in pain behavior remains poorly investigated. Therefore, the present study evaluated whether the presence of reactive glia (hypertrophy, increased number and upregulation of glial markers) in the ventral posterolateral thalamic nucleus (VPL) correlates with pain symptoms, 14 and 28 days after unilateral L5/L6 spinal nerve ligation (SNL) in rats.MethodsMechanical allodynia and hyperalgesia (von Frey filament stimulation) as well as ambulatory pain (dynamic weight bearing apparatus) were assessed. Levels of nine glial transcripts were determined by quantitative real-time PCR on laser microdissected thalamic nuclei, and levels of proteins were assessed by Western blot. We also studied by immunohistofluorescence the expression of glial markers that label processes (GFAP for astrocytes and iba-1 for microglia) and cell body (S100β for astrocytes and iba-1 for microglia) and quantified the immunostained surface and the number of astrocytes and microglia (conventional counts and optical dissector method of stereological counting).ResultsDifferential, time-dependent responses were observed concerning microglia and astrocytes. Specifically, at day 14, iba-1 immunostained area and number of iba-1 immunopositive cells were decreased in the VPL of SNL as compared to naïve rats. By contrast, at day 28, GFAP-immunostained area was increased in the VPL of SNL as compared to naïve rats while number of GFAP/S100β immunopositive cells remained unchanged. Using quantitative real-time PCR of laser microdissected VPL, we found a sequential increase in mRNA expression of cathepsin S (day 14), fractalkine (day 28), and fractalkine receptor (day 14), three well-known markers of microglial reactivity. Using Western blot, we confirmed an increase in protein expression of fractalkine receptor at day 14.ConclusionsOur results demonstrate a sequential alteration of microglia and astrocytes in the thalamus of animals with lesioned peripheral nerves. Furthermore, our data report unprecedented concomitant molecular signs of microglial activation and morphological signs of microglial decline in the thalamus of these animals.
Highlights
Spinal reactive astrocytes and microglia are known to participate to the initiation and maintenance of neuropathic pain
We studied the mRNA expression of the genes coding for three proteins that are expressed in microglia and the expression of which has been shown to be altered in reactive microglia: iba-1 [36, 37], the cluster of differentiation molecule 11 b (CD11b) [38, 39], and the fractalkine receptor (CX3CR1) [40,41,42,43]
spinal nerve ligation (SNL) curves displayed a shift to the left between day 0 (D0) and day 14 (D14) and between D0 and day 28 (D28): previously non painful stimuli became painful, a process known as allodynia
Summary
Spinal reactive astrocytes and microglia are known to participate to the initiation and maintenance of neuropathic pain. Astrocytes become hypertrophied (more numerous and thicker processes as well as an enlarged cell body) with an increased expression of glial acidic fibrillary protein (GFAP) and S100β for instance [2,3,4,5,6,7,8,9,10] while microglial cells become hypertrophied and have increased ionized calcium binding adaptor molecule 1 (iba-1) expression [7, 11,12,13] Under these morphological changes, astrocytes and microglia have been referred to as reactive or activated. Spinal astrocytes play a role in the maintenance of peripheral nerve injury-induced pain symptoms [10, 22]
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