Number of intraepithelial lymphocytes and presence of a subepithelial band in normal colonic mucosa differs according to stainings and evaluation method.

Chronic watery diarrhea is a frequent symptom. In approximately 10% of the patients, a diagnosis of microscopic colitis (MC) is established. The diagnosis relies on specific, but sometimes subtle, histopathological findings. As the histology of normal intestinal mucosa vary, discriminating subtle features of MC from normal tissue can be challenging and therefore auxiliary stainings are increasingly used. The aim of this study was to determine the variance in number of intraepithelial lymphocytes (IELs) and presence of a subepithelial band in normal ileum and colonic mucosa, according to different stains and digital assessment. Sixty-one patients without diarrhea referred to screening colonoscopy due to a positive feacal blood test and presenting with endoscopically normal mucosa were included. Basic histological features, number of IELs, and thickness of a subepithelial band was manually evaluated and a deep learning-based algorithm was developed to digitally determine the number of IELs in each of the two compartments; surface epithelium and cryptal epithelium, and the density of lymphocytes in the lamina propria compartment. The number of IELs was significantly higher on CD3-stained slides compared with slides stained with Hematoxylin-and-Eosin (HE) (p<0.001), and even higher numbers were reached using digital analysis. No significant difference between right and left colon in IELs or density of CD3-positive lymphocytes in lamina propria was found. No subepithelial band was present in HE-stained slides while a thin band was visualized on special stains. Conclusively, in this cohort of prospectively collected ileum and colonic biopsies from asymptomatic patients, the range of IELs and detection of a subepithelial collagenous band varied depending on the stain and method used for assessment. As assessment of biopsies from patients with diarrhea constitute a considerable workload in the pathology departments digital image analysis is highly desired. Knowledge provided by the present study highlight important differences that should be considered before introducing this method in the clinic.

Immunohistochemical Staining With Neuroendocrine Markers is Essential in the Diagnosis of Neuroendocrine Neoplasms of the Esophagogastric Junction.

Neuroendocrine neoplasms (NENs) of the esophagogastric junction (EGJ) are uncommon and the classification of these tumors has been revised several times. Since 2016, at the Department of Pathology, Rigshospitalet, Denmark, all adenocarcinomas and poorly differentiated carcinomas of the EGJ have been stained routinely with the neuroendocrine markers, synaptophysin and chromogranin A, to detect a possible neuroendocrine component. This study aimed to determine if routine immunohistochemical staining is necessary to detect neuroendocrine differentiation of the EGJ tumors by evaluating how often a neuroendocrine component of the tumors was correctly identified or missed on routine hematoxylin and eosin-stained slides, and by evaluating the interobserver agreement among several pathologists. Of 262 cases a NEN was identified in 24 (9.2%). Up to 22.7% of all EGJ NENs would have been missed without routinely performed neuroendocrine staining in all EGJ tumors. The interobserver agreement between 3 pathologists was slight to moderate. In conclusion, immunohistochemical staining with neuroendocrine markers is essential for the diagnosis of NENs, and to detect all NENs, we recommend to perform this routinely on all resected tumors of the EGJ.

Establishment of digital cutoff values for intraepithelial lymphocytes in biopsies from colonic mucosa with lymphocytic colitis.

BACKGROUND/INTRODUCTION: Lymphocytic colitis (LC) and the incomplete form (LCi) are common causes of chronic watery diarrhea. Endoscopy is often inconspicuous, and the diagnosis relies on histopathological assessment of colonic biopsies. Digital Image Analysis (DIA) eliminates interobserver variation. The aim of this study was to establish digital cutoff values for LC and LCi on CD3 stained slides. MATERIAL AND METHODS: One hundred and six patients with a hematoxylin and eosin (HE) diagnosis of normal colonic mucosa (N = 19), non-specific reactive changes (N = 24), LCi (N = 23) and LC (N = 40) were eligible for analysis. The number of intraepithelial lymphocytes (IELs) reached by DIA in the total surface epithelium and in hot spots of the biopsies was compared with the diagnostic category assigned by the pathologists based on HE stained slides. The digitalized slides were analyzed for number of IELs using Visiopharm Quantitative Digital Pathology software. All digitalized slides were examined manually to identify differences in the approach to the evaluation of the biopsies by the pathologists and DIA. RESULTS: The median IEL counts and interquartile range in the total surface epithelium were 3.6 (3.2-4.3), 4.4 (3.4-5.3), 19.8 (16.6-30.0) and 41.3 (37.0-47.8) in normal colon mucosa, mucosa with non-specific reactive changes, LCi and LC, respectively. Discrimination between normal mucosa and non-specific reactive changes was not possible. Digital cutoff values with the best separation between non-LC, LCi and LC were > 13 IELs/100 epithelial cells for LCi and > 36 IELs/100 epithelial cells for LC. These cutoff values resulted in an agreement between the pathologist's and DIA that was very good with a kappa value of 0.90. CONCLUSION: Despite differences among the approach of DIA and the pathologist's assessment of IELs in colonic mucosa DIA is able discriminate between the HE based diagnoses of the three subgroups non-LC, LCi and LC with high accuracy.

Is revision of cutoff values needed when using CD3 immunohistochemical staining in histopathologic diagnosis of lymphocytic colitis?

Lymphocytic colitis (LC) and LC incomplete (LCi) are common causes of chronic watery diarrhea. The diagnosis relies on clinical findings and histopathologic evaluation. The diagnostic criteria of LC are based on hematoxylin and eosin (HE) staining. However, supplementary immunohistochemical staining for highlighting the lymphocytes in borderline cases is now widely used. This change in diagnostics could lead to incorrectly diagnosing patients with LC and LCi if the present histologic criteria are used. The number of intraepithelial lymphocytes (IELs) was estimated and categorized in intervals based on HE- versus CD3-stained slides from patients with an HE diagnosis of normal colonic mucosa (n = 19), mucosa with nonspecific reactive changes (n = 24), LCi (n = 24), and LC (n = 40). The number of IELs was compared with clinical symptoms. Overall, the number of IELs was higher with CD3 stain compared with HE stain in 73% of cases, unchanged in 26% of cases, and lower in 1 case. The number of IELs detected was higher using the CD3 stain in 53%, 79%, 79%, and 75% of cases included as normal colonic mucosa, nonspecific reactive changes, LCi, and LC, respectively. Based on CD3 stain, 58% of the cases with nonspecific reactive changes fulfilled the HE criteria for LCi, and 79% of the cases with LCi fulfilled the HE criteria for LC. Automated image analysis of CD3-stained slides resulted in even higher numbers of IELs in all 4 diagnostic groups. Conclusively, our data support considering increased cutoff values for LCi and LC when assessed in CD3-stained specimens.

Automated image analysis in the study of collagenous colitis.

PURPOSE: The aim of this study was to develop an automated image analysis software to measure the thickness of the subepithelial collagenous band in colon biopsies with collagenous colitis (CC) and incomplete CC (CCi). The software measures the thickness of the collagenous band on microscopic slides stained with Van Gieson (VG). PATIENTS AND METHODS: A training set consisting of ten biopsies diagnosed as CC, CCi, and normal colon mucosa was used to develop the automated image analysis (VG app) to match the assessment by a pathologist. The study set consisted of biopsies from 75 patients. Twenty-five cases were primarily diagnosed as CC, 25 as CCi, and 25 as normal or near-normal colonic mucosa. Four pathologists individually reassessed the biopsies and categorized all into one of the abovementioned three categories. The result of the VG app was correlated with the diagnosis provided by the four pathologists. RESULTS: The interobserver agreement for each pair of pathologists ranged from κ-values of 0.56-0.81, while the κ-value for the VG app vs each of the pathologists varied from 0.63 to 0.79. The overall agreement between the four pathologists was κ=0.69, while the overall agreement between the four pathologists and the VG app was κ=0.71. CONCLUSION: In conclusion, the Visiopharm VG app is able to measure the thickness of a sub-epithelial collagenous band in colon biopsies with an accuracy comparable to the performance of a pathologist and thereby provides a promising supplementary tool for the diagnosis of CC and CCi and in particular for research.

CD3 immunohistochemical staining in diagnosis of lymphocytic colitis.

Microscopic colitis (MC) is a common cause of chronic watery diarrhea. Traditionally, MC encompasses the 2 subgroups lymphocytic colitis (LC) and collagenous colitis, but recently, an additional subgroup, MC incomplete, has been introduced. Distinguishing between the subgroups relies exclusively on histopathologic evaluation. In the present study, 4 pathologists evaluated 156 archived biopsies originally diagnosed as LC or LC incomplete (LCi). Each pathologist assigned a diagnosis of LC, LCi, or nonspecific inflammation to all cases at 2 independent assessments. At the first assessment, hematoxylin and eosin (HE) stainings were available. At the second assessment, a supplementary CD3 immunohistochemical staining was also available. The aim was to evaluate whether a supplementary CD3 would increase the diagnostic agreement among pathologists, and whether a CD3 stain would change the diagnosis based on HE staining only. After the complete assessment, the cases were divided into 3 groups, that is, full agreement, partial agreement, and disagreement. The CD3 staining increased the number of cases with full agreement from 60 to 78. One hundred thirty-one cases with agreement or partial diagnostic agreement based on HE + CD3 were compared with the HE diagnoses. In 44 (34%) of 131 cases, CD3 changed the diagnosis. Cases assigned to the LCi category based on HE were often changed by a supplementary CD3. Conclusively, it is recommended to use a CD3 before giving the histopathologic diagnosis of LCi.

Involvement of CDX2 in the cross talk between TNF-α and Wnt signaling pathway in the colon cancer cell line Caco-2.

Tumor necrosis factor-α (TNF-α) is highly upregulated in inflammation and reduces the expression of the intestinal transcription factor, Caudal-related homeobox transcription factor 2 (CDX2). Wnt/ß-catenin signaling is critical for intestinal cell proliferation, but a decreased CDX2 expression has influence on the Wnt signaling-related genes and progression of colorectal cancer. Although several inflammatory signaling pathways, including TNF-α, have been reported to promote Wnt/ß-catenin activity and development of cancer, the underlying molecular mechanisms remain unclear. The aim was to investigate the signaling pathways involved in the TNF-α-mediated downregulation of CDX2, and its influence on Wnt/ß-catenin signaling components in colon cancer cells. The expression of TNF-α and CDX2 at the invasive front were evaluated by immunohistochemical staining and showed reduced CDX2-positive cells in tumor buddings in areas with TNF-α expression in the surrounding inflammatory cells. In vitro studies revealed that TNF-α treatment showed a dose-dependent decrease of CDX2 messenger RNA (mRNA) and protein expression in Caco-2 cells. Inhibition of nuclear factor-kappaB or p38 pathways showed that these are involved in the TNF-α-dependent downregulation of CDX2. Furthermore, TNF-α-mediated downregulation of CDX2 was found to significantly decrease the mRNA levels of adenomatous polyposis coli (APC), axis inhibition protein 2 (AXIN2) and glycogen synthase kinase-3 beta (GSK3ß), whereas the mRNA levels of Wnt targets were significantly elevated in TNF-α-treated Caco-2 cells. These findings were associated with reduced binding of CDX2 to promoter or enhancer regions of APC, AXIN2 and GSK3ß. In conclusion, it was found that TNF-α induces the expression of Wnt signaling components through a downregulation of the CDX2 expression that might have a tumor-promoting effect on colon cancer cells.

Is the longitudinal margin of carcinoma-bearing colon resections a neglected parameter?

BACKGROUND: Resection of colon cancer with curative intent implies clear margins. An arbitrary requirement of 2 cm DtLM generally ensures surgical and pathological clearance. However, harvest of tumor-draining lymph nodes is related to DtLM. For this reason, an extended longitudinal margin becomes an issue. The major objective of the present study concerns quality development of colon resections, recording the status of DtLM, pT and pN stage, and the pathologists' reporting pattern. MATERIALS AND METHODS: The study comprised colectomy specimens obtained in 2010 to 2011 at Hvidovre Hospital with documented and suspected carcinoma. Specimens were stratified into 2 groups: DtLM < 5 cm and ≥ 5 cm. Data were correlated with lesional site, surgical approach, pT and pN stage and the pathologists' reporting approach. RESULTS: DtLM reporting was lacking in 6% of the specimens. DtLM was < 5 cm in 32% of the specimens. Sixty-three and 83.5% of the cancer specimens with DtLM < 5 cm were node-negative and stage pT3/4, respectively, compared with 49% and 87.5% of the ≥ 5 cm counterpart. The difference in percentage distribution of pN stage in the 2 groups was significant, and no significant difference was observed in relation to pT stage. CONCLUSION: This study suggests that DtLM < 5 cm in colon cancer surgery might result in diagnostic "understaging" and hence leaving metastasis in the patient.

Regulation of APC and AXIN2 expression by intestinal tumor suppressor CDX2 in colon cancer cells.

Wnt signaling is often constitutively active in colorectal cancer cells. The expression of the intestinal specific transcription factor CDX2 is found to be transiently decreased in invasive cells at the tumor/stroma interface. A recent ChIP-Seq study has indicated that several Wnt signaling-related genes are regulated by CDX2. The aim was to investigate the role of decreased CDX2 level on the expression of APC, AXIN2 and GSK3ß in migrating colon cancer cells at the invasive front. CDX2-bound promoter and enhancer regions from APC, AXIN2 and GSK3ß were analyzed for gene regulatory activity and the expression pattern of APC and GSK3ß at the invasive front was evaluated by immunohistochemical procedures. Transfection of intestinal and non-intestinal cell lines demonstrated that CDX2 activated APC and AXIN2 promoter activities via intestinal cell-specific enhancer elements. Suppressed CDX2 expression was associated with endogenous downregulation of APC and AXIN2 expression in Caco-2 cells but did not affect GSK3ß expression. Furthermore, elevated levels of nuclear ß-catenin and reduced levels of cytoplasmic APC were correlated to a low CDX2 expression in migrating colon cancer cells in vivo. These results suggest that a low CDX2 level has influence on the Wnt signaling in invasive colon cancer cells possibly promoting cellular migration.

Significance of acquired diverticular disease of the vermiform appendix: a marker of regional neoplasms?

AIM: To assess the prevalence of acquired diverticulum of the appendix (DA), including incipient forms and its possible significance as a marker of local/regional neoplasms. MATERIALS AND METHODS: The pathology database at Hvidovre Hospital was searched for appendix specimens, received between 2001 and 2010, coded for DA or for a space-occupying lesion. Slides were reviewed to determine DA status and the nature of lesions possibly causing DA. RESULT: Among 4413 appendix specimens, DA were identified in 39 (0.9%, CI 0.6% to 1.2%) cases, 17 (43.6%, 28.0% to 59.2%) of which additionally harboured an appendiceal neoplasm/neoplastic precursor, whereas this figure was 1.2% (CI 0.9% to 1.6%) for non-DA specimens (p<0.0001). Six of the 39 DA specimens comprised incipient DA, three of which coexisted with appendiceal neoplasms. In addition, local/regional non-neoplastic lesions (six cases) and colorectal carcinomas (four cases) coexisted with DA. CONCLUSION: DA has significance as a putative marker of local/regional neoplasms. Therefore, a DA specimen proved significantly more likely to harbour a neoplastic growth than a non-DA counterpart. Submission for microscopy of the entire DA specimen, whether transmural or only incipient, and a comment in the pathology report on the occasional concurrence of local/regional neoplasms in this setting seem appropriate. The observation of DA may thus provide a valuable contribution in the diagnostic process.

[Tumour-cell dissociation as a prognostic marker in colorectal cancer]. / Tumorcelledissociering som prognostisk markør ved kolorektal cancer.

The invasive front of the colorectal carcinoma (CRC) in some cases displays budding, characterized by groups of up to five tumour cells. In 2006, budding was introduced in the Danish Colorectal Cancer Group's (DCCG) and the Danish Society of Pathology and Cytology's (DSPAC) CRC register form. Based on a literature survey, molecular mechanisms that may contribute to budding are reviewed, as is the mode by which this process is registered and its putative clinical significance. Despite diverse modes of budding registration, its significance as a prognostic marker has consistently been substantiated. Additionally, data on budding may prove of value in patient management.

[Lymph node identification in colorectal cancer specimens cases]. / Fund af lymfeknuder ved kolorektal cancer.

Colorectal carcinoma is one of the most prevalent malignancies in Western countries. Lymph node status is a significant prognosticator. The chance of identifying node-positivity is positively correlated with the number of lymph nodes (LN) identified. The present paper discusses various variables that may influence the detection of LNs, including patient- as well as surgeon- and pathologist-related issues. The pathologist-related variable most probably shapes the yield the most. Introduction of guidelines focusing on the most appropriate technique may secure better and more consistent results, and the pathologist's commitment is crucial in this respect.

[Resection time and number of detected colorectal lymph nodes in resection specimens with carcinoma]. / Udskaeringstid og antal påviste lymfeknuder ved kolorektal cancer.

INTRODUCTION: The number of identified lymph nodes (LNs) is an essential element in the pathologist's rapport on colorectal resection specimens with carcinoma (CRSC). A considerable number of papers discuss the acceptable minimum number of identified LNs to secure a correct LN status (LNS). Details as to the most appropriate grossing technique for LN detection are, however, largely lacking. In this paper the influence of the time invested by the pathologist in the pursuit of LN is investigated. MATERIAL AND METHODS: The material comprised 150 CRSCs. The usual gross examination was extended by 15 minutes in an effort to identify additional LNs. Provided this careful analysis failed to produce 12 LNs and all detected LNs were benign (pNx), the specimen was re-sampled for an additional 15-minute period. Data were correlated with a baseline material comprising 100 CRSCs. RESULTS: The intensified search for LNs increased the average number of LNs pr. specimen from 9.1 to 14.9. The number of cases with pNx was reduced from 54% to 18%. Re-sampling performed on 25 specimens resulted in the detection of another 61 LNs in 21 cases, ranging from 1 to 8 LN pr. specimen (median 2), whereby pNx was converted to pN0 in eight cases. In another four cases, additional LNs were not detected. Re-sampling did not uncover metastatic disease. CONCLUSION: This intensified effort in the Department of Pathology resulted in a more reliable LNS.

Biopsies of colorectal clinical polyps--emergence of diagnostic information on deeper levels.

Although the occasional appearance of a normal histology of biopsies from endoscopic colorectal (CR) polyps is generally held knowledge, its prevalence has rarely been focused on, and the yield of additional sections in such cases has been previously addressed in merely four communications. Hitherto, this issue has not been discussed in the context of the clinical setting. The prime aim of this study was to evaluate the yield of step sectioning CR biopsies, considered non-diagnostic (non-diagnostic biopsies (NDB)) on routine sections. The results are correlated with the indications for endoscopy. Additionally, an appropriate, cost-effective approach for handling NDB was sought. Biopsies from 480 clinical polyps were prospectively evaluated by one of three gastrointestinal pathologists and classified as (a) diagnostic biopsies (DB), comprising neoplastic polyps, hyperplastic polyps (HP), sessile-serrated polyp, other diverse causes of polyp formation and (b) NDB comprising normal histology (group 1), suspicious of either adenoma (group 2) or HP (group 3). Material grouped 1-3 was subsequently step-sectioned (three sections prepared from each of nine additional levels). The biopsy specimens were obtained from 245 endoscopies and stratified in the following categories according to the clinical indications: relevant symptoms (symptomatic, n=127), previously documented sporadic large bowel neoplasia (follow-up, n=99), and documented or presumed hereditary condition that confer an increased risk of CRC (hereditary, n=19, including 15 hereditary non-polyposis colorectal cancer (HNPCC) cases). Sixty-five (13.5%) of the 480 samples were classified as NDB (normal morphology n=49, suspicious of adenoma n=5, suspicious of HP n=11), constituting roughly 10% of all biopsies from the symptomatic and the follow-up categories, 32.1% of samples from the hereditary cases, the difference between the hereditary and the non-hereditary cases being statistically significant (p<0.0001). Upon leveling the 65 NDB, a DB emerged in 24 (36.9%) cases, with no significant difference in the yield in relation to the delineated indication categories. Thereby, diagnostic information was obtained with three additional levels in 15 cases, the remaining 9 cases requiring additional sections, ranging from 4 to 8 levels. The present step sectioning approach implied an extra expense of about 112 US$ for each NDB converted to a DB. The higher prevalence of NDB in relation to genetic disorders probably reflects sampling of particularly diminutive lesions. Given the high yield of step sectioning NDB coupled with an acceptable price, our strategy delineated here is recommended in routine practice with the modification of an initial preparation of sections from merely three levels, and if still non-diagnostic, supplementation with additional five levels.

Cervical vertebrae, cranial base, and mandibular retrognathia in human triploid fetuses.

On profile radiographs of adults, an association between fusions of cervical vertebrae, deviations in the cranial base and mandibular retrognathia has been documented radiographically. An elaboration of this association on a histological level is needed. In human triploid fetuses severe mandibular retrognathia and deviations in the cranial base have previously been described radiographically (without cephalometry) and cervical column fusions radiographically as well as histologically. Therefore, triploid fetuses were chosen to elucidate the cranial base cephalomterically and histologically. In the present study, eight triploid fetuses were analyzed radiographically and histologically focusing especially on the cranial base, which borders to the spine and to which the jaws are attached. A histological analysis of the cranial base has not previously been performed in triploid cases. An enlarged cranial base angle and a retrognathic position of the mandible were documented cephalometrically on radiographs of all cases. Histologically, malformations were observed in the cranial base as well as in the spine. These are new findings indicating the association between the occipital bone and the uppermost vertebra in the body axis. As the notochord connects the cervical column and the cranial base in early prenatal life, molecular signaling from the notochord may in future studies support the notochord as the developmental link between abnormal development in the spine and the cranial base.

Phosphodiesterase 5 and effects of sildenafil on cerebral arteries of man and guinea pig.

Sildenafil (Viagra), a selective inhibitor of phosphodiesterase 5 (PDE5), induces headache and migraine. Although previously supposed to be a "vascular" headache, no significant cerebral artery dilatation was found in vivo. Thus, we hypothesised that PDE5 may not be present or that sildenafil is less effective on the cGMP hydrolysis in cerebral arteries, and that sildenafil may not be an effective dilator of cerebral arteries under baseline conditions. We evaluated the presence of PDE5 mRNA and protein in human arteries. Furthermore, the effects of two selective PDE5 inhibitors, sildenafil and UK-114,542, and a PDE1 inhibitor UK-90,234 on cGMP hydrolysis were investigated in human and guinea pig cerebral arteries. The vasoactive responses of the compounds were evaluated in guinea pig basilar arteries in vitro, with concomitant measurements of cAMP and cGMP. PDE5 was found in human middle cerebral arteries. Sildenafil and UK-114,542 inhibited cGMP hydrolysis concentration-dependently in both species. In guinea pig arteries, sildenafil induced an endothelium-dependent vasodilatation only at concentrations above 10 nM, which was augmented by sodium nitroprusside and attenuated by reduction of cGMP, but was cGMP independent at high concentrations. UK-114,542 was more and UK-90,234 was less potent than sildenafil. In conclusion, PDE5 is present in human and guinea pig cerebral arteries, and is inhibited by sildenafil at micromolar levels. Sildenafil in vitro is a poor dilator of guinea pig cerebral arteries unless a nitric oxide donor is co-administered, corresponding to the previous findings in vivo.

Trisomy 10 mosaicism and maternal uniparental disomy 10 in a liveborn infant with severe congenital malformations.

We report on a liveborn infant with trisomy 10 mosaicism combined with maternal uniparental heterodisomy for chromosome 10. The mosaicism 47,XY,+10/46,XY was found in five different tissues, including one blood sample, while cultured lymphocytes from two other blood samples showed a normal karyotype, 46,XY. DNA analysis with six PCR-based microsatellite markers demonstrated the trisomic cell line to be a result of maternal meiotic nondisjunction, and revealed maternal uniparental heterodisomy in the diploid cell line, suggesting that the formation of the diploid cell line was due to trisomy rescue. The boy had severe growth retardation, major dysmorphism, and malformations, and died at 37 days. We reviewed the previous nine reports of infants and fetuses with trisomy 10 mosaicism reported in the literature. We suggest that a common clinical syndrome can be defined comprising skull, jaw and ear abnormalities, cleft lip/palate, malformations of eyes, heart and kidneys, deformity of hands and feet, and most often death neonatally or in early infancy. The cytogenetic findings in the present patient demonstrate the importance of karyotyping more than one tissue, and not only lymphocytes, when a chromosomal aberration is strongly suspected.

Peptidergic and non-peptidergic innervation and vasomotor responses of human lenticulostriate and posterior cerebral arteries.

The aim of the present study was to compare in man the innervation pattern and the functional responses to neuronal messengers in medium sized lenticulostriate and branches of the posterior cerebral arteries (PCA). The majority of the nerve fibers found were sympathetic and displayed specific immunoreactivity for tyrosine hydroxylase (TH) and neuropeptide Y (NPY). Only few nerve fibers displayed vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and substance P (SP) immunoreactivity. In both arteries, the contractions induced by noradrenaline (NA), NPY and 5-hydroxytryptamine (5-HT) and the relaxant responses induced by acetylcholine (ACh), VIP and pituitary adenylate cyclase activating peptide-27 (PACAP) as well as CGRP and SP were compared in vitro. In conclusion, there was no major difference in innervation pattern or vasomotor sensitivity (pEC50 and pIC50 values) between the two vessels. However, the general pattern indicates stronger vasomotor responses (Emax and Imax) in the PCA branches as compared to the lenticulostriate arteries which may lend support for the clinical observation of a difference in stroke expression between the two vascular areas.

In-depth characterization of CGRP receptors in human intracranial arteries.

The purpose of the present study was to characterize the effects of human (h) alpha- and beta-calcitonin gene-related peptide (CGRP) on intracranial arteries from man and to investigate the presence of mRNA for the calcitonin receptor like receptor (CRLR) and the receptor activity modifying proteins (RAMPs) 1, 2 and 3, in cerebral and middle meningeal arteries with and without endothelium, in microvessels and in the endothelial cells isolated from the human basilar artery. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of CRLR, RAMP 1, RAMP 2 and RAMP 3 in cerebral and middle meningeal arteries with and without endothelium as well as in microvessels and in the endothelial cells. Human and rat alpha- and beta-CGRP, amylin, adrenomedullin and [acetamidomethyl-Cys(2,7)]human CGRP induced strong concentration-dependent relaxation of human cerebral and middle meningeal arteries. Removal of the endothelium neither changed the maximum relaxant response nor the pIC(50) values for alpha- and beta-CGRP as compared to the responses in arteries with an intact endothelium. Human alpha-CGRP-(8-37) caused a shift of h alpha- and h beta-CGRP-induced relaxations in cerebral and middle meningeal arteries. Calculation of pK(B) values revealed that h alpha-CGRP-(8-37) could not significantly discriminate between relaxations induced by h alpha-CGRP (pK(B) around 6.8) and h beta-CGRP (pK(B) around 5.4). There was no significant difference in pK(B) value of h alpha-CGRP-(8-37) on h beta-CGRP-induced relaxation of human cerebral and middle meningeal arteries with and without endothelium. In conclusion, our molecular and pharmacological data support the existence of a single type of CGRP(1) receptors in the human intracranial circulation.