Immunohistochemical visualization of neurons and specific glial cells for stereological application in the porcine neocortex

Lyck, Lise; Jelsing, Jacob; Jensen, Peter Bjødstrup; Lambertsen, Kate Lykke; Pakkenberg, Bente; Finsen, Bente

doi:10.1016/j.jneumeth.2005.09.009

Cited by 24 publications

(30 citation statements)

References 39 publications

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“…Parallel control experiments double labelling for sGC and a non-neuronal cell (astroglia: marker GFAP) were also performed. The cellular staining pattern for GFAP was consistent with the morphology and distribution of astroglia [40], and showed no colocalisation or pattern similarity to the sGC staining (Fig. 4b).…”

Section: Detection Of Sgc-ir In the Rvlmsupporting

confidence: 54%

Immunohistochemical assessment of cyclic guanosine monophosphate (cGMP) and soluble guanylate cyclase (sGC) within the rostral ventrolateral medulla

et al. 2008

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2008).Immunohistochemical assessment of cyclic guanosine monophosphate (cGMP) and soluble guanylate cyclase (sGC) within the rostral ventrolateral medulla. Journal of Biomedical Science, 15 (6), 801-812. Immunohistochemical assessment of cyclic guanosine monophosphate (cGMP) and soluble guanylate cyclase (sGC) within the rostral ventrolateral medulla AbstractFunctional evidence suggests that nitric oxide (NO) signalling in the rostral ventrolateral medulla (RVLM) is cGMP-dependent and that this pathway is impaired in hypertension. We examined cGMP expression as a marker of active NO signalling in the C1 region of the RVLM, comparing adult (>18 weeks) Wistar-Kyoto (WKY, n = 4) and spontaneously hypertensive rats (SHR, n = 4). Double label immunohistochemistry for cGMP-immunoreactivity (IR) and C1 neurons [as identified by phenylethanolamine N-methyltransferase (PNMT-IR) or tyrosine hydroxylase TH-IR)], or neuronal NO synthase (nNOS) neurones, failed to reveal cGMP-IR neurons in the RVLM of either strain, despite consistent detection of cGMP-IR in the nucleus ambiguus (NA). This was unchanged in the presence of isobutylmethylxanthine (IBMX; 0.5 mM, WKY, n = 4, SHR n = 2) and in young animals (WKY, 10-weeks, n = 3). Incubation of RVLM-slices (WKY, 10-weeks, n = 9) in DETA-NO (100 μm; 10 min) or NMDA (10 μM; 2 min) did not uncover cGMP-IR. In all studies, cGMP was prominent within the vasculature. Soluble guanylate cyclase (sGC)-IR was found throughout neurones of the RVLM, but did not co-localise with PNMT, TH or nNOS-IR neurons (WKY, 10-weeks, n = 6). Results indicate that within the RVLM, cGMP is not detectable using immunohistochemistry in the basal state and cannot be elicited by phosphodiesterase inhibition, NMDA receptor stimulation or NO donor application. Abstract Functional evidence suggests that nitric oxide (NO) signalling in the rostral ventrolateral medulla (RVLM) is cGMP-dependent and that this pathway is impaired in hypertension. We examined cGMP expression as a marker of active NO signalling in the C1 region of the RVLM, comparing adult ([18 weeks) Wistar-Kyoto (WKY, n = 4) and spontaneously hypertensive rats (SHR, n = 4). Double label immunohistochemistry for cGMPimmunoreactivity (IR) and C1 neurons [as identified by phenylethanolamine N-methyltransferase (PNMT-IR) or tyrosine hydroxylase TH-IR)], or neuronal NO synthase (nNOS) neurones, failed to reveal cGMP-IR neurons in the RVLM of either strain, despite consistent detection of cGMP-IR in the nucleus ambiguus (NA). This was unchanged in the presence of isobutylmethylxanthine (IBMX; 0.5 mM, WKY, n = 4, SHR n = 2) and in young animals (WKY, 10-weeks, n = 3). Incubation of RVLMslices (WKY, 10-weeks, n = 9) in DETA-NO (100 lm; 10 min) or NMDA (10 lM; 2 min) did not uncover cGMP-IR. In all studies, cGMP was prominent within the vasculature. Soluble guanylate cyclase (sGC)-IR was found throughout neurones of the RVLM, but did not co-localise with PNMT, TH or nNOS-IR neurons (WKY, 10-weeks, n = 6). Results indicate that within the RVLM, cGMP is not ...

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Section: Detection Of Sgc-ir In the Rvlmsupporting

confidence: 54%

Immunohistochemical assessment of cyclic guanosine monophosphate (cGMP) and soluble guanylate cyclase (sGC) within the rostral ventrolateral medulla

et al. 2008

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“…The lack of clear criteria for distinguishing neurons and glial cells in cortical regions has previously been a major problem for stereologists (Braendgaard et al, 1990;Davanlou and Smith, 2004) and may partly explain the somewhat lower hemispheric cell number published in a screening procedure of young Göttingen minipig (Jelsing et al, 2005a). Recently, a number of immunohistochemical markers have been evaluated in the pig brain (Lyck et al, 2006), and the combination of immunohistochemistry and stereology may provide a better approach for quantifying neurons and glial cell populations in future quantitative studies of the pig brain.…”

Section: Stereological Designmentioning

confidence: 99%

The postnatal development of neocortical neurons and glial cells in the Göttingen minipig and the domestic pig brain

Jelsing

Nielsen

Olsen

et al. 2006

Journal of Experimental Biology

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SUMMARY The first mathematically unbiased estimates of neocortical cell numbers are presented from the developing pig brain, including a full description of tissue processing and optimal sampling for application of the stereological optical fractionator method in this species. The postnatal development of neocortical neurons and glial cells from the experimental Göttingen minipig was compared with the postnatal development of neocortical neurons in the domestic pig. A significant postnatal development was observed in the Göttingen minipig brain for both neuronal (28%; P=0.01) and glial cells (87%; P<0.01). A corresponding postnatal development of neurons was not detected in the domestic pig brain. The reason for this strain difference is not known. The mean total number of neocortical neurons is 324 million in the adult Göttingen minipig compared with 432 million in the domestic pig. The glial-to-neuron cell ratio is around 2.2 in the adult Göttingen minipig. Based on these results, the domestic pig seems to be a more suitable model for evaluating the effects of developmental insults on human brain growth and neuronal development than the Göttingen minipig.

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“…In order to determine if E2 is neuroprotective in the medial amygdala (MeA), we used neuronal marker neuron-specific protein (NeuN) to label brain sections of OVX vehicle- and E2-treated animals [E2-treated animals received different regimens, including our previously studied high dose of E2 (10 µg for 14 days)] and we counted the immunolabeled neurons using stereological methods. We selected this neuronal marker because of its demonstrated applicability to stereology [25]. Using densitometric methods in our previous studies, we have shown an effect of E2 (10 µg for 14 days ) on pCREB levels [2, 5].…”

Section: Introductionmentioning

confidence: 99%

Dose and Time Effects of Estrogen on Expression of Neuron-Specific Protein and Cyclic AMP Response Element-Binding Protein and Brain Region Volume in the Medial Amygdala of Ovariectomized Rats

Liu

Hanbury

Pandey³

et al. 2008

Neuroendocrinology

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Although estrogen has been shown to be neuroprotective, studies concerning its effect on some behaviors are contradictory, reporting both ameliorative and detrimental effects. A factor involved in hormone efficacy is the estrogen regimen. We reported an effect of 10 µg estrogen for 14 days on the cyclic AMP response element-binding protein (CREB) pathway, including brain-derived neurotrophic factor, in rat medial amygdala (MeA). To determine the effects of estrogen on neuronal numbers and brain region volume in MeA and central nucleus of the amygdala (CeA), we used stereology to test the effect of various estrogen regimens on the number of neuron-specific protein (NeuN)-labeled neurons and brain region volume of MeA and CeA. Ovariectomized rats were injected with vehicle for 14 days, 2.5 µg estradiol benzoate (E2) for 4 or 14 days, or 10 µg estrogen for 14 days. Because NeuN-labeled neuronal number may be related to neuronal survival and upregulation of CREB signaling, we tested the effect of these regimens on levels of phosphorylated CREB (pCREB) labeling in the MeA and CeA. The 2.5 µg estrogen for 14 days regimen increased the mean number of NeuN-labeled neurons and pCREB-labeled cells in the MeA compared to vehicle or 2.5 µg for 4 days. There was an increase in volume of the MeA with 2.5 µg estrogen for 14 days compared to vehicle or 2.5 µg for 4 days. No differences in these parameters were seen in CeA. These data indicate a neuroanatomical heterogeneity of a time effect of estrogen on cells expressing NeuN and pCREB in the MeA versus CeA.

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Immunohistochemical visualization of neurons and specific glial cells for stereological application in the porcine neocortex

Cited by 24 publications

References 39 publications

Immunohistochemical assessment of cyclic guanosine monophosphate (cGMP) and soluble guanylate cyclase (sGC) within the rostral ventrolateral medulla

Immunohistochemical assessment of cyclic guanosine monophosphate (cGMP) and soluble guanylate cyclase (sGC) within the rostral ventrolateral medulla

The postnatal development of neocortical neurons and glial cells in the Göttingen minipig and the domestic pig brain

Dose and Time Effects of Estrogen on Expression of Neuron-Specific Protein and Cyclic AMP Response Element-Binding Protein and Brain Region Volume in the Medial Amygdala of Ovariectomized Rats

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