2D <em>vs</em> 3D morphological analysis of dorsal root ganglia in health and painful neuropathy

Dorsal root ganglia (DRGs) are clusters of sensory neurons that transmit the sensory information from the periphery to the central nervous system, and satellite glial cells (SGCs), their supporting trophic cells. Sensory neurons are pseudounipolar neurons with a heterogeneous neurochemistry reflecting their functional features. DRGs, not protected by the blood brain barrier, are vulnerable to stress and damage of different origin (i.e., toxic, mechanical, metabolic, genetic) that can involve sensory neurons, SGCs or, considering their intimate intercommunication, both cell populations. DRG damage, primary or secondary to nerve damage, produces a sensory peripheral neuropathy, characterized by neurophysiological abnormalities, numbness, paraesthesia and dysesthesia, tingling and burning sensations and neuropathic pain. DRG stress can be morphologically detected by light and electron microscope analysis with alterations in cell size (swelling/atrophy) and in different sub-cellular compartments (i.e., mitochondria, endoplasmic reticulum, and nucleus) of neurons and/or SGCs. In addition, neurochemical changes can be used to portray abnormalities of neurons and SGC. Conventional immunostaining, i.e., immunohistochemical detection of specific molecules in tissue slices can be employed to detect, localize and quantify particular markers of damage in neurons (i.e., nuclear expression ATF3) or SGCs (i.e., increased expression of GFAP), markers of apoptosis (i.e., caspases), markers of mitochondrial suffering and oxidative stress (i.e., 8-OHdG), markers of tissue inflammation (i.e., CD68 for macrophage infiltration), etc. However classical (2D) methods of immunostaining disrupt the overall organization of the DRG, thus resulting in the loss of some crucial information. Whole-mount (3D) methods have been recently developed to investigate DRG morphology and neurochemistry without tissue slicing, giving the opportunity to study the intimate relationship between SGCs and sensory neurons in health and disease. Here, we aim to compare classical (2D) vs whole-mount (3D) approaches to highlight “pros” and “cons” of the two methodologies when analysing neuropathy-induced alterations in DRGs.

Download Full-text

Generation of New Neurons in Dorsal Root Ganglia in Adult Rats after Peripheral Nerve Crush Injury

Neural Plasticity ◽

10.1155/2015/860546 ◽

2015 ◽

Vol 2015 ◽

pp. 1-12 ◽

Cited By ~ 11

Author(s):

Luisa Muratori ◽

Giulia Ronchi ◽

Stefania Raimondo ◽

Stefano Geuna ◽

Maria Giuseppina Giacobini-Robecchi ◽

...

Keyword(s):

Sensory Neurons ◽

Dorsal Root Ganglia ◽

Dorsal Root ◽

Morphological Changes ◽

Dorsal Root Ganglion Neurons ◽

Cell Phenotype ◽

Adult Rats ◽

Nerve Crush ◽

Satellite Glial Cells ◽

Ganglion Neurons

The evidence of neurons generatedex novoin sensory ganglia of adult animals is still debated. In the present study, we investigated, using high resolution light microscopy and stereological analysis, the changes in the number of neurons in dorsal root ganglia after 30 days from a crush lesion of the rat brachial plexus terminal branches. Results showed, as expected, a relevant hypertrophy of dorsal root ganglion neurons. In addition, we reported, for the first time in the literature, that neuronal hypertrophy was accompanied by massive neuronal hyperplasia leading to a 42% increase of the number of primary sensory neurons. Moreover, ultrastructural analyses on sensory neurons showed that there was not a relevant neuronal loss as a consequence of the nerve injury. The evidence of BrdU-immunopositive neurons and neural progenitors labeled with Ki67, nanog, nestin, and sox-2 confirmed the stereological evidence of posttraumatic neurogenesis in dorsal root ganglia. Analysis of morphological changes following axonal damage in addition to immunofluorescence characterization of cell phenotype suggested that the neuronal precursors which give rise to the newly generated neurons could be represented by satellite glial cells that actively proliferate after the lesion and are able to differentiate toward the neuronal lineage.

Download Full-text

An update on the spinal and peripheral pathways of pain signalling

e-Neuroforum ◽

10.1515/nf-2017-a020 ◽

2017 ◽

Vol 23 (3) ◽

Cited By ~ 1

Author(s):

Stefan G. Lechner

Keyword(s):

Spinal Cord ◽

Sensory Neurons ◽

Dorsal Root Ganglia ◽

Dorsal Root ◽

Neural Circuits ◽

Sensory Information ◽

Brain Regions ◽

Sensory Afferents ◽

Different Types ◽

Functionally Diverse

AbstractPainful or potentially tissue-damaging stimuli are detected by primary sensory afferents that innervate the skin as well as internal tissues. The neurons that give rise to sensory afferents are located in the dorsal root ganglia (DRG) and transmit sensory information to the spinal cord where it is processed and further relayed to higher brain regions to ultimately generate the perception of pain. Both the DRGs as well as the spinal cord comprise a variety of morphologically, molecularly and functionally diverse neurons. The objective of this review is to provide an overview of the different types of sensory neurons and their proposed role in pain signalling. Moreover, I will discuss how pain related sensory information is processed in the dorsal horn of the spinal cord with an emphasis on recently delineated neural circuits that mediate pain hypersensitivity in the setting of nerve injury and inflammation.

Download Full-text

Painful Nerve Injury Decreases Resting Cytosolic Calcium Concentrations in Sensory Neurons of Rats

Anesthesiology ◽

10.1097/00000542-200506000-00023 ◽

2005 ◽

Vol 102 (6) ◽

pp. 1217-1225 ◽

Cited By ~ 33

Author(s):

Andreas Fuchs ◽

Philipp Lirk ◽

Cheryl Stucky ◽

Stephen E. Abram ◽

Quinn H. Hogan

Keyword(s):

Nerve Injury ◽

Sensory Neurons ◽

Dorsal Root Ganglia ◽

Dorsal Root ◽

Sensory Information ◽

Cytosolic Calcium ◽

Skin Incision ◽

Cellular Level ◽

Spinal Nerve ◽

Control Group

Background Neuropathic pain is difficult to treat and poorly understood at the cellular level. Although cytoplasmic calcium ([Ca]c) critically regulates neuronal function, the effects of peripheral nerve injury on resting sensory neuronal [Ca]c are unknown. Methods Resting [Ca]c was determined by microfluorometry in Fura-2 AM-loaded neurons dissociated from dorsal root ganglia of animals with hyperalgesia to mechanical stimulation after spinal nerve ligation and section (SNL) at the fifth and sixth lumbar (L5 and L6) levels and from animals after skin incision alone (control group). Axotomized neurons from the L5 dorsal root ganglia were examined separately from adjacent L4 neurons that share the sciatic nerve with degenerating L5 fibers. Results After SNL, large (34 mum or larger) neurons from the L4 ganglion showed a 29% decrease in resting [Ca]c, whereas those from the L5 ganglion showed a 54% decrease. Small neurons only showed an effect of injury in the axotomized L5 neurons, in which resting [Ca]c decreased by 30%. A decrease in resting [Ca]c was not seen in neurons isolated from rats in which hyperalgesia did not develop after SNL. In separate experiments, SNL reduced resting [Ca]c in capsaicin-insensitive neurons of the L5 ganglion by 60%, but there was no change in neurons from L4. Resting [Ca]c of capsaicin-sensitive neurons was not affected by injury in either ganglion. SNL injury decreased the proportion of neurons sensitive to capsaicin in the L5 group but increased the proportion in the L4 group. Conclusions Painful SNL nerve injury depresses resting [Ca]c in sensory neurons. This is most marked in axotomized neurons, especially the large and capsaicin-insensitive neurons presumed to transmit non-nociceptive sensory information.

Download Full-text

The 5-HT2A receptor is mainly expressed in nociceptive sensory neurons in rat lumbar dorsal root ganglia

Neuroscience ◽

10.1016/j.neuroscience.2009.03.087 ◽

2009 ◽

Vol 161 (3) ◽

pp. 838-846 ◽

Cited By ~ 33

Author(s):

J. Van Steenwinckel ◽

A. Noghero ◽

K. Thibault ◽

M.-J. Brisorgueil ◽

J. Fischer ◽

...

Keyword(s):

Sensory Neurons ◽

Dorsal Root Ganglia ◽

Dorsal Root

Download Full-text

M2-Receptor Subtype Does Not Mediate Muscarine-Induced Increases in [Ca2+]i in Nociceptive Neurons of Rat Dorsal Root Ganglia

Journal of Neurophysiology ◽

10.1152/jn.2000.84.4.1934 ◽

2000 ◽

Vol 84 (4) ◽

pp. 1934-1941 ◽

Cited By ~ 21

Author(s):

Rainer Haberberger ◽

Reas Scholz ◽

Wolfgang Kummer ◽

Michaela Kress

Keyword(s):

Receptor Antagonist ◽

Muscarinic Receptor ◽

Sensory Neurons ◽

Dorsal Root Ganglia ◽

Dorsal Root ◽

Receptor Subtype ◽

Receptor Subtypes ◽

Muscarinic Receptor Subtypes ◽

Rt Pcr ◽

Nociceptive Neurons

Multiple muscarinic receptor subtypes are present on sensory neurons that may be involved in the modulation of nociception. In this study we focused on the presence of the muscarinic receptor subtypes, M2 and M3 (M2R, M3R), in adult rat lumbar dorsal root ganglia (DRG) at the functional ([Ca2+]i measurement), transcriptional (RT-PCR), and translational level (immunohistochemistry). After 1 day in culture exposure of dissociated medium-sized neurons (20–35 μm diam) to muscarine was followed by rises in [Ca2+]i in 76% of the neurons. The [Ca2+]i increase was absent after removal of extracellular calcium and did not desensitize after repetitive application of the agonist. This rise in [Ca2+]i may be explained by the expression of M3R, which can induce release of calcium from internal stores via inositoltrisphospate. Indeed the effect was antagonized by the muscarinic receptor antagonist atropine as well as by the M3R antagonist, 4-diphenylacetoxy-N-(2 chloroethyl)-piperidine hydrochloride (4-DAMP). The pharmacological identification of M3R was corroborated by RT-PCR of total RNA and single-cell RT-PCR, which revealed the presence of mRNA for M3R in lumbar DRG and in single sensory neurons. In addition, RT-PCR also revealed the expression of M2R, which did not seem to contribute to the calcium changes since it was not prevented by the M2 receptor antagonist, gallamine. Immunohistochemistry demonstrated the presence of M2R and M3R in medium-sized lumbar DRG neurons that also coexpressed binding sites for the lectin I-B4, a marker for mainly cutaneous nociceptors. The occurrence of muscarinic receptors in putative nociceptive I-B4-positive neurons suggests the involvement of these acetylcholine receptors in the modulation of processing of nociceptive stimuli.

Download Full-text

Morphological and electrophysiological changes in mouse dorsal root ganglia after partial colonic obstruction

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00028.2005 ◽

2005 ◽

Vol 289 (4) ◽

pp. G670-G678 ◽

Cited By ~ 36

Author(s):

Tian-Ying Huang ◽

Menachem Hanani

Keyword(s):

Glial Cells ◽

Dorsal Root Ganglia ◽

Dorsal Root ◽

Neuronal Excitability ◽

Colonic Obstruction ◽

Resting Potential ◽

Dye Coupling ◽

Drg Neurons ◽

Satellite Glial Cells ◽

Colon Wall

There is evidence that sensitization of neurons in dorsal root ganglia (DRG) may contribute to pain induced by intestinal injury. We hypothesized that obstruction-induced pain is related to changes in DRG neurons and satellite glial cells (SGCs). In this study, partial colonic obstruction was induced by ligation. The neurons projecting to the colon were traced by an injection of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate into the colon wall. The electrophysiological properties of DRG neurons were determined using intracellular electrodes. Dye coupling was examined with an intracellular injection of Lucifer yellow (LY). Morphological changes in the colon and DRG were examined. Pain was assessed with von Frey hairs. Partial colonic obstruction caused the following changes. First, coupling between SGCs enveloping different neurons increased 18-fold when LY was injected into SGCs near neurons projecting to the colon. Second, neurons were not coupled to other neurons or SGCs. Third, the firing threshold of neurons projecting to the colon decreased by more than 40% ( P < 0.01), and the resting potential was more positive by 4–6 mV ( P < 0.05). Finally, the number of neurons displaying spontaneous spikes increased eightfold, and the number of neurons with subthreshold voltage oscillations increased over threefold. These changes are consistent with augmented neuronal excitability. The pain threshold to abdominal stimulation decreased by 70.2%. Inflammatory responses were found in the colon wall. We conclude that obstruction increased neuronal excitability, which is likely to be a major factor in the pain behavior observed. The augmented dye coupling between glial cells may contribute to the neuronal hyperexcitability.

Download Full-text

Purinergic signaling between neurons and satellite glial cells of mouse dorsal root ganglia modulates neuronal excitability in vivo

Pain ◽

10.1097/j.pain.0000000000002556 ◽

2021 ◽

Vol Publish Ahead of Print ◽

Author(s):

Zhiyong Chen ◽

Qian Huang ◽

Xiaodan Song ◽

Neil C. Ford ◽

Chi Zhang ◽

...

Keyword(s):

Glial Cells ◽

Dorsal Root Ganglia ◽

Dorsal Root ◽

Neuronal Excitability ◽

Purinergic Signaling ◽

Satellite Glial Cells

Download Full-text

Frataxin gene editing rescues Friedreich’s ataxia pathology in dorsal root ganglia organoid-derived sensory neurons

Nature Communications ◽

10.1038/s41467-020-17954-3 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Pietro Giuseppe Mazzara ◽

Sharon Muggeo ◽

Mirko Luoni ◽

Luca Massimino ◽

Mattia Zaghi ◽

...

Keyword(s):

Sensory Neurons ◽

Dorsal Root Ganglia ◽

Dorsal Root ◽

Gene Editing ◽

Friedreich’S Ataxia ◽

Friedreich's Ataxia ◽

Frataxin Gene

Download Full-text

RNA-Binding Proteins HuB, HuC, and HuD are Distinctly Regulated in Dorsal Root Ganglia Neurons from STZ-Sensitive Compared to STZ-Resistant Diabetic Mice

International Journal of Molecular Sciences ◽

10.3390/ijms20081965 ◽

2019 ◽

Vol 20 (8) ◽

pp. 1965 ◽

Cited By ~ 1

Author(s):

Cosmin Cătălin Mustăciosu ◽

Adela Banciu ◽

Călin Mircea Rusu ◽

Daniel Dumitru Banciu ◽

Diana Savu ◽

...

Keyword(s):

Gene Expression ◽

Body Weight ◽

Sensory Neurons ◽

Dorsal Root Ganglia ◽

Dorsal Root ◽

Transcriptional Control ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Diabetic Mice

The neuron-specific Elav-like Hu RNA-binding proteins were described to play an important role in neuronal differentiation and plasticity by ensuring the post-transcriptional control of RNAs encoding for various proteins. Although Elav-like Hu proteins alterations were reported in diabetes or neuropathy, little is known about the regulation of neuron-specific Elav-like Hu RNA-binding proteins in sensory neurons of dorsal root ganglia (DRG) due to the diabetic condition. The goal of our study was to analyze the gene and protein expression of HuB, HuC, and HuD in DRG sensory neurons in diabetes. The diabetic condition was induced in CD-1 adult male mice with single-intraperitoneal injection of streptozotocin (STZ, 150 mg/kg), and 8-weeks (advanced diabetes) after induction was quantified the Elav-like proteins expression. Based on the glycemia values, we identified two types of responses to STZ, and mice were classified in STZ-resistant (diabetic resistant, glycemia < 260 mg/dL) and STZ-sensitive (diabetic, glycemia > 260 mg/dL). Body weight measurements indicated that 8-weeks after STZ-induction of diabetes, control mice have a higher increase in body weight compared to the diabetic and diabetic resistant mice. Moreover, after 8-weeks, diabetic mice (19.52 ± 3.52 s) have longer paw withdrawal latencies in the hot-plate test than diabetic resistant (11.36 ± 1.92 s) and control (11.03 ± 1.97 s) mice, that correlates with the installation of warm hypoalgesia due to the diabetic condition. Further on, we evidenced the decrease of Elav-like gene expression in DRG neurons of diabetic mice (Elavl2, 0.68 ± 0.05 fold; Elavl3, 0.65 ± 0.01 fold; Elavl4, 0.53 ± 0.07 fold) and diabetic resistant mice (Ealvl2, 0.56 ± 0.07 fold; Elavl3, 0.32 ± 0.09 fold) compared to control mice. Interestingly, Elav-like genes have a more accentuated downregulation in diabetic resistant than in diabetic mice, although hypoalgesia was evidenced only in diabetic mice. The Elav-like gene expression changes do not always correlate with the Hu protein expression changes. To detail, HuB is upregulated and HuD is downregulated in diabetic mice, while HuB, HuC, and HuD are downregulated in diabetic resistant mice compared to control mice. To resume, we demonstrated HuD downregulation and HuB upregulation in DRG sensory neurons induced by diabetes, which might be correlated with altered post-transcriptional control of RNAs involved in the regulation of thermal hypoalgesia condition caused by the advanced diabetic neuropathy.

Download Full-text