Transcriptomic Profiling of Lactotroph Pituitary Neuroendocrine Tumors via RNA Sequencing and Ingenuity Pathway Analysis.

INTRODUCTION: Lactotroph pituitary neuroendocrine tumors (PitNETs) are common pituitary tumors, but their underlying molecular mechanisms remain unclear. This study aimed to investigate the transcriptomic landscape of lactotroph PitNETs and identify potential molecular mechanisms and therapeutic targets through RNA sequencing and ingenuity pathway analysis (IPA). METHODS: Lactotroph PitNET tissues from five surgical cases without dopamine agonist treatment underwent RNA sequencing. Normal pituitary tissues from three patients served as controls. Differentially expressed genes (DEGs) were identified, and the functional pathways and gene networks were explored by IPA. RESULTS: Transcriptome analysis revealed that lactotroph PitNETs had gene expression patterns that were distinct from normal pituitary tissues. We identified 1,172 upregulated DEGs, including nine long intergenic noncoding RNAs (lincRNAs) belonging to the top 30 DEGs. IPA of the upregulated DEGs showed that the estrogen receptor signaling, oxidative phosphorylation signaling, and EIF signaling were activated. In gene network analysis, key upstream regulators, such as EGR1, PRKACA, PITX2, CREB1, and JUND, may play critical roles in lactotroph PitNETs. CONCLUSION: This study provides a comprehensive transcriptomic profile of lactotroph PitNETs and highlights the potential involvement of lincRNAs and specific signaling pathways in tumor pathogenesis. The identified upstream regulators may be potential therapeutic targets for future investigations.

Lung adenocarcinoma metastasis within a pituitary neuroendocrine tumor: a case report with review of literature.

Collision tumors involving the metastasis of malignant neoplasms to pituitary neuroendocrine tumors (PitNETs) are extremely rare. We herein report a case involving a patient with lung adenocarcinoma metastasis within a PitNET who exhibited relatively rapid progression of neurological symptoms. A 75-year-old man who underwent tumor resection 36 and 18 years prior to presentation for bladder and colon cancer, respectively, without recurrence presented with bitemporal hemianopsia, ptosis, and diplopia of the right eye. Subsequent magnetic resonance imaging (MRI) revealed a tumor 3.2 cm in diameter that extended from the anterior pituitary gland to the suprasellar region. Gadolinium-enhanced MRI of the tumor showed heterogeneous contrast enhancement. Considering the relatively rapid progression of neurological symptoms, semi-emergency endoscopic endonasal transsphenoidal surgery was performed. Histopathological examination revealed a group of thyroid transcription factor-1- and napsin A-positive papillary proliferating cells intermingled with α-subunit- and steroidogenic factor-1-positive PitNET cells. Thus, the patient was diagnosed with lung adenocarcinoma metastasis within a gonadotroph PitNET. Genetic testing revealed the presence of an EGFR (Ex-19del) mutation, after which chemotherapy was initiated. Additional stereotactic radiotherapy was performed for the residual tumor in the sella turcica. With continued chemotherapy, good control of both the primary and metastatic tumors was noted after 24 months after surgery. Cases of malignant neoplasm metastasis within a PitNET are difficult to diagnose. In the case of a sella turcica tumor with relatively rapid progression of neurological symptoms, early surgical intervention is recommended given the possibility of a highly proliferative tumor and the need to obtain pathologic specimens.

Endoscopic spinal cord untethering using a 1 cm skin incision technique in pediatrics: a technical case report.

BACKGROUND: Spinal cord untethering by sectioning the filum terminale is commonly performed in tethered cord syndrome patients with minor abnormalities such as filar lipoma, thickened filum terminale, and low conus medullaris. Our endoscopic surgical technique, using the interlaminar approach, allows for sectioning the filum terminale through a very small skin incision. To our knowledge, this procedure has not been previously reported. This is the first case report involving a 1 cm skin incision. CASE PRESENTATION: A 9-month-old male patient was referred to our neurosurgical department due to a coccygeal dimple. MRI revealed a thickened fatty filum. After considering the treatment options for this patient, the parents agreed to spinal cord untethering. A midline 1 cm skin incision was made at the L4/5 vertebral level. Untethering by sectioning the filum terminale was performed by full endoscopic surgery using the interlaminar approach. The procedure was uneventful and there were no postoperative complications. CONCLUSIONS: In terms of visibility and minimizing invasiveness, our surgical technique of using the interlaminar approach with endoscopy allows for untethering by sectioning the filum terminale through a very small skin incision.

Comparison of prophylactic antibiotics for endonasal transsphenoidal surgery using a national inpatient database in Japan.

BACKGROUND: The choice of prophylactic antibiotics for use in endonasal transsphenoidal surgery (ETSS) lacks universal standards. This study aimed to investigate the effectiveness of cefazolin, ampicillin and third-generation cephalosporins for preventing postoperative meningitis and secondary outcomes (in-hospital death and the combination of pneumonia and urinary tract infection) in patients who have undergone ETSS. METHODS: The study used data from the Diagnosis Procedure Combination database in Japan. Data from 10 688 patients who underwent ETSS between April 2016 and March 2021 were included. Matching weight analysis based on propensity scores was conducted to compare the outcomes of patients receiving cefazolin, ampicillin or third-generation cephalosporins as prophylactic antibiotics. RESULTS: Of the 10 688 patients, 9013, 102 and 1573 received cefazolin, ampicillin and third-generation cephalosporins, respectively. The incidence of postoperative meningitis did not significantly differ between the cefazolin group and the ampicillin group (OR, 1.02; 95% CI, 0.14-7.43) or third-generation cephalosporins group (OR, 0.81; 95% CI, 0.10-6.44). Similarly, in-hospital death and the composite incidence of pneumonia and urinary tract infection did not differ between the cefazolin group and the ampicillin or third-generation cephalosporins group. CONCLUSIONS: Cefazolin, ampicillin and third-generation cephalosporins as perioperative prophylactic antibiotics for ETSS do not differ significantly in terms of preventing meningitis.

Endoscopic surgery versus craniotomy for spontaneous intracerebral hemorrhage in the late elderly patients.

OBJECTIVES: To compare the prognosis of late elderly patients with spontaneous intracerebral hemorrhage (ICH) treated by endoscopic evacuation and craniotomy MATERIALS AND METHODS: Using the Diagnosis Procedure Combination database, we identified patients aged ≥ 75 years who underwent surgery for spontaneous ICH within 48 hours after admission between April 2014 and March 2018. Eligible patients were classified into two groups according to the type of surgery (endoscopic surgery and craniotomy). Propensity-score matching weight analysis was conducted to compare the good neurological outcome modified Rankin Scale (mRS) score (0-4) at discharge as the primary endpoint between the two groups. Secondary endpoints were postoperative meningitis, tracheostomy, reoperation within 3 days and total hospitalization costs. RESULTS: Among the 5,396 eligible patients, endoscopic surgery and craniotomy were performed in 895 and 4,501 patients, respectively. In the propensity-score matching weight analysis, all covariates were well balanced. The proportions of patients with a good prognosis (mRS score at discharge: 0-4) did not significantly differ between the surgical procedures (42.1% vs. 42.8%, p = 0.828). The proportions of meningitis, tracheostomy and reoperation were not significantly different between the two groups. Hospitalization costs were significantly higher in the craniotomy group than in the endoscopic surgery group (25,536 vs. 29,603 US dollars, p = 0.012). CONCLUSIONS: Inhospital outcomes did not differ between endoscopic and open surgeries for spontaneous ICH in the late-stage elderly patients aged ≥75 years. Hospitalization costs were significantly higher in the craniotomy group, suggesting that endoscopic surgery may be more acceptable.

Estrogen Receptor α Isoforms Generated by Alternative Use of Cryptic Exons.

Estrogen receptor α (ERα) regulates several physiological functions. In pathophysiological conditions, ERα is involved in the development and progression of estrogen-sensitive tumors. The ERα gene contains multiple 5'-untranslated exons and eight conventional coding exons and presents multiple isoforms generated by alternative promoter usage and alternative splicing. This gene also possesses non-conventional exons in the 3'- and intronic regions, and alternative use of cryptic exons contributes to further diversity of ERα mRNAs and proteins. Recently, the genomic organization of ERα genes and the splicing profiles of their transcripts were comparatively analyzed in humans, mice, and rats, and multiple ERα isoforms with distinct structures and functions were identified. These transcripts contain cryptic sequences that encode insertion-containing or truncated ERα proteins. In particular, alternative cryptic exons with in-frame stop codons yield transcripts encoding C-terminally-truncated ERα proteins. The C-terminally-truncated ERα isoforms lack part or all of the ligand-binding domain but possess an isoform-specific sequence. Some of these isoforms exhibit constitutive transactivation and resistance to estrogen receptor antagonists. Although numerous studies have reported conflicting results regarding their functions, the critical determinant for their gain-of-function has been identified structurally. Here we review recent progress in ERα variant research concerning the genomic organization of ERα genes, splicing profiles of ERα transcripts, and transactivation properties of ERα isoforms.

Exploring endocrinological pitfalls in pituitary surgery in the elderly.

Background: Endoscopic transsphenoidal surgery (ETSS) is performed more frequently in elderly patients. We investigated endocrinological pitfalls in pituitary surgery in the elderly by a comparative study focusing only on elderly patients. Methods: Ninety-nine elderly patients aged 65 years and over with non-functioning pituitary adenoma (NFPA) who underwent ETSS were retrospectively examined and classified into the early (aged 65-74 years) and late (aged 75 years and over) elderly groups. The baseline characteristics and anterior pituitary function were compared between the groups. Results: Seventy patients were assigned to the early elderly group and 29 to the late elderly group. Thyroid-stimulating hormone (TSH) response in preoperative and postoperative thyrotropin-releasing hormone (TRH) tests revealed a significant difference between the groups. Preoperative and postoperative TSH responses were significantly correlated in both groups. Residual analysis of the correlation between preoperative free triiodothyronine (T3) secretion quantity and preoperative TSH response in both groups, which was significant, indicated that preoperative TSH response was significantly normal when preoperative free T3 secretion quantity was normal in the early elderly group, but preoperative free T3 secretion quantity was significantly lower regardless of preoperative TSH response in the late elderly group. Conculsions: The present study suggested that preoperative and postoperative TSH secretory capacity was presumed to be normal when preoperative free T3 levels were normal in the early elderly patients with NFPA. On the other hand, TSH secretory capacity in the late elderly patients could only be assessed by the TRH test, which should be taken into account.

Assessment of anterior pituitary reserve capacity based on growth hormone response to growth hormone-releasing peptide-2 test in the elderly.

OBJECTIVE: The growth hormone (GH)-releasing peptide-2 (GHRP-2) test is relatively safe among endocrine stimulation tests for the elderly. We investigated whether anterior pituitary function in elderly patients could be assessed on the basis of GH response to the GHRP-2 test. DESIGN: Sixty-five elderly patients aged 65 years and older with non-functioning pituitary neuroendocrine tumor (PitNET) who underwent pituitary surgery and preoperative endocrine stimulation tests were classified into the "GH normal group" and "GH deficiency group" based on GH response to the GHRP-2 test. The baseline characteristics and anterior pituitary function were compared between the groups. RESULTS: Thirty-two patients were assigned to the GH normal group and 33 to the GH deficiency group. The cortisol and adrenocorticotropic hormone (ACTH) results in the corticotropin-releasing hormone test were significantly higher in the GH normal group than in the GH deficiency group (p < 0.001). The relationship between the cortisol and ACTH results and the GH response revealed significant correlations (p < 0.001). In addition, receiver operating characteristic curve analysis identified that the optimal cut-off point for a peak GH level in the correlation between adrenocortical function and GH response to the GHRP-2 test was 8.08 ng/mL (specificity 0.868, sensitivity 0.852). CONCLUSION: The present study indicated that adrenocortical function was significantly correlated with GH response to the GHRP-2 test in elderly patients before pituitary surgery. For elderly patients with non-functioning PitNET, GH response to the GHRP-2 test may support in diagnosing adrenocortical insufficiency.

Concurrent IgG4-related hypophysitis and clinically nonfunctioning gonadotroph pituitary neuroendocrine tumor.

BACKGROUND: Some patients develop immunoglobulin G4 (IgG4)-related hypophysitis associated with systemic diseases. More than 30 cases of IgG4-related hypophysitis have been reported. However, biopsy has rarely been performed in these patients, and none have had an associated pituitary neuroendocrine tumor (PitNET). We present a case of concurrent IgG4-related hypophysitis and PitNET. CASE PRESENTATION: A 56-year-old Japanese man arrived at the hospital with visual impairment, bitemporal hemianopia, and right abducens nerve palsy. Magnetic resonance imaging revealed pituitary body and stalk swelling as well as a small poorly enhanced right anterior lobe mass. Laboratory and loading test results suggested hypopituitarism. Because IgG4 level was elevated, a systemic examination was performed; multiple nodules were found in both lung fields. The diagnosis was based on an endoscopic transnasal biopsy of the pituitary gland. A histopathological examination revealed a marked infiltration of plasma cells into the pituitary gland, which was strongly positive for IgG4. The histological features of the resected tumor were consistent with those of gonadotroph PitNET, which was immunohistochemically positive for follicle-stimulating hormone-ß and steroidogenic factor-1, and no plasma cell infiltration was observed. Based on the histopathological examination results, steroid therapy was initiated, which reduced pituitary gland size and serum IgG4 levels. DISCUSSION AND CONCLUSIONS: This is the first reported case of IgG4-related hypophysitis with PitNET. Although no pathological findings indicating a relationship between the two conditions were found, we were able to preoperatively differentiate multiple lesions via detailed diagnostic imaging.

Creation of low cost, simple, and easy-to-use training kit for the dura mater suturing in endoscopic transnasal pituitary/skull base surgery.

Training kits for laparoscopes for deep suturing under endoscopes are commercially available; however, previously reported training kits for endoscopic transnasal transsphenoidal pituitary/skull base surgery (eTSS) were not available in the market. Moreover, the previously reported low cost, self-made kit has the drawback of being unrealistic. This study aimed to create a low cost training kit for eTSS dura mater suturing that was as close to real as possible. Most necessary items were obtained from the 100-yen store ($1 store) or from everyday supplies. As an alternative to the endoscope, a stick-type camera was used. Through the assembly of the materials, a simple and easy-to-use training kit was created, which is almost identical to the actual dural suturing situation. In eTSS, a simple and easy-to-use training kit for dural suturing was successfully created at a low cost. This kit is expected to be used for deep suture operations and the development of surgical instruments for training.

Outcomes After Endoscopic Evacuation Versus Evacuation Using Craniotomy or Stereotactic Aspiration for Spontaneous Intracerebral Hemorrhage: Analysis Using a Japanese Nationwide Database.

BACKGROUND: Various surgical methods are available for managing large intracerebral hemorrhage. This study compared the prognosis of patients with spontaneous intracerebral hemorrhage who underwent endoscopic evacuation, stereotactic aspiration, and craniotomy by using a nationwide inpatient database in Japan. METHODS: Using the Diagnosis Procedure Combination database, we identified patients who underwent surgery for spontaneous intracerebral hemorrhage within 48 h after admission between April 2014 and March 2018. Eligible patients were classified into three groups according to the type of surgery (endoscopic surgery, stereotactic surgery, and craniotomy). Propensity score matching weight analysis was conducted to compare poor modified Rankin Scale score at discharge (severe disability or death) and hospitalization cost among the groups. RESULTS: Among 17,860 eligible patients, craniotomy, stereotactic surgery, and endoscopic surgery were performed in 14,354, 474, and 3,032 patients, respectively. In the matching weight analysis, all covariates were well balanced. Compared with the endoscopic surgery group, the proportion of poor prognosis (modified Rankin Scale score at discharge of 5 or 6) was significantly higher in craniotomy groups (odds ratio 2.51, 95% confidence interval 1.11-5.68; p = 0.028). Subgroup analysis based on hemorrhage location and consciousness level at the time of admission showed no significant difference between the surgical procedures. Hospitalization costs were significantly higher in the craniotomy group than in the endoscopic surgery group (difference US $9,724, 95% confidence interval 2,169-17,259; p = 0.011). CONCLUSIONS: Endoscopic surgery for spontaneous intracerebral hemorrhage was associated with improved prognosis compared with craniotomy at the hospital discharge. Future large-scale clinical trials are needed to evaluate the optimal surgical techniques for intracerebral hemorrhage.

Optimized Mouse-on-mouse Immunohistochemical Detection of Mouse ESR2 Proteins with PPZ0506 Monoclonal Antibody.

Despite the physiological significance of ESR2, a lack of well-validated detection systems for ESR2 proteins has hindered progress in ESR2 research. Thus, recent identification of a specific anti-human ESR2 monoclonal antibody (PPZ0506) and its specific cross-reactivity against mouse and rat ESR2 proteins heightened momenta toward development of appropriate immunohistochemical detection systems for rodent ESR2 proteins. Building upon our previous optimization of ESR2 immunohistochemical detection in rats using PPZ0506, in this study, we further aimed to optimize mouse-on-mouse immunohistochemical detection using PPZ0506. Our assessment of several staining conditions using paraffin-embedded ovary sections revealed that intense heat-induced antigen retrieval, appropriate blocking, and appropriate antibody dilutions were necessary for optimization of mouse-on-mouse immunohistochemistry. Subsequently, we applied the optimized immunostaining method to determine expression profiles of mouse ESR2 proteins in peripheral tissues and brain subregions. Our analyses revealed more localized distribution of mouse ESR2 proteins than previously assumed. Moreover, comparison of these results with those obtained in humans and rats using PPZ0506 revealed interspecies differences in ESR2 expression. We expect that our optimized methodology for immunohistochemical staining of mouse ESR2 proteins will help researchers to solve multiple lines of controversial evidence concerning ESR2 expression.

An Overview of Pituitary Incidentalomas: Diagnosis, Clinical Features, and Management.

Pituitary incidentalomas are tumors or mass lesions of the pituitary gland. These are incidentally discovered during imaging studies for symptoms that are not causally related to pituitary diseases. The most common symptom that triggers an examination is headache, and the most common type of pituitary incidentalomas are pituitary neuroendocrine tumors (PitNETs) and Rathke cleft cysts. The existing treatment strategy is controversial; however, surgical resection is recommended in cases of clinically non-functioning PitNETs with optic chiasm compression. In contrast, cystic lesions, such as Rathke cleft cysts, should be followed if the patients are asymptomatic. In this case, MRI and pituitary function tests are recommended every six months to one year; if there is no change, the follow-up period should be extended. The natural history of PitNET is partially known, and the management of pituitary incidentalomas is determined by this history. However, the pathogenesis of PitNET has significantly changed with the new World Health Organization classification, and follow-up is important based on this new classification. Therefore, a high level of evidence-based research is needed to consider treatment guidelines for pituitary incidentalomas in the future.

Trends in surgical procedures for spontaneous intracerebral hemorrhage in Japan.

OBJECTIVES: Minimally invasive surgery for spontaneous intracerebral hemorrhage (ICH) has become increasingly popular in recent years. However, there are no reports on the recent trends in surgical procedures for spontaneous ICH. To investigate current trends in surgical methods for spontaneous ICH using a nationwide inpatient database from Japan. MATERIALS AND METHODS: Patients who underwent surgery for spontaneous ICH between April 2014 and March 2018 were identified in a nationwide inpatient database from Japan. We examined patient characteristics, diagnoses, types of surgery, complications, and discharge status. RESULTS: We identified 21,129 inpatients who underwent surgery for spontaneous ICH. The procedures were as follows: 16,256 (76.9%) transcranial hemorrhage evacuations, 3722 (17.6%) endoscopic hemorrhage evacuations, and 1151 (5.4%) stereotactic aspirations of hemorrhage. Patients tended to receive transcranial hemorrhage evacuations in hospitals with fewer surgical cases. The proportions of endoscopic hemorrhage evacuations increased annually, whereas those of stereotactic surgery decreased. The proportions of transcranial surgery remained almost unchanged. Tracheostomy and hospitalization costs were lower in the stereotactic aspirations of hemorrhage group, and the proportions of reoperation were higher in the endoscopic hemorrhage evacuations group. CONCLUSIONS: The use of endoscopic surgery for spontaneous ICH has increased in Japan. This study can form the basis of future clinical investigations into spontaneous ICH surgery.

Injury to the Extrasellar Portion of the Internal Carotid Artery during Endoscopic Transsphenoidal Surgery: A Case Report.

Background: Injury to the internal carotid artery (ICA) during endoscopic transsphenoidal surgery (ETSS) is a serious complication with a risk of mortality. ICA injury during ETSS usually occurs during intrasellar manipulations and rarely occurs in the extrasellar portion. Several hemostatic procedures have been proposed for ICA injury in the intrasellar portion, whereas hemostatic methods for ICA injury in the extrasellar portion, where the ICA is surrounded by bone structures, are less well known. Case Presentation: A 65-year-old man with an incidental pituitary tumor underwent ETSS. The petrous portion of the left ICA was injured during resection of the sphenoid septum connected with left carotid prominence using a cutting forceps. Bleeding was too heavy for simple hemostatic techniques. Hemostasis using a crushed muscle patch was tried unsuccessfully during controlling of the bleeding. Eventually, the injured site of the ICA was covered with cotton patties followed by closing with a vascularized pedicled nasoseptal flap. Cerebral angiography immediately after surgery showed no extravasation from the injured site of the left ICA petrous portion. However, a carotid-cavernous sinus fistula originating from the injured ICA site was detected 7 days after surgery, so the vascular reconstructive surgery combined with left ICA occlusion was performed. The overall postoperative course was uneventful. Conclusion: We believe that emergency application of the cottonoids may be effective for hemostasis against ICA injury in the extrasellar portion during ETSS, but further vascular reconstruction combined with ICA occlusion on the injured side and removal of the cottonoids would be required.

Bacterial Flora in the Sphenoid Sinus Changes with Perioperative Prophylactic Antibiotic Administration.

The complications of endonasal transsphenoidal surgery (ETSS) include meningitis and sinusitis, and these complications are troublesome. Some reports have investigated the type of bacteria and the susceptibility of sphenoid sinus mucosal flora to drugs. However, most specimens can be collected after perioperative antibiotic administration. In this study, 95 and 103 sphenoid sinus mucosal samples collected during ETSS from September 2013 to February 2015 and from June 2017 to January 2019, respectively, were examined for bacterial culture. Sphenoid sinus mucosal samples were collected after antibiotic administration in the first period, whereas samples were collected before antibiotic administration in the second period. Hence, the specimens in the second period were not affected by antibiotics. Moreover, drug susceptibility tests for the detected bacteria were performed. Overall, 52 and 51 bacterial isolates were collected during both periods. Gram-positive cocci (GPCs), including Staphylococcus aureus and Staphylococcus epidermidis, were more common in the non-antibiotic group than in the antibiotic group (p <0.01). However, the proportion of gram-negative rods (GNRs) did not significantly differ between the two groups (p = 0.54). The antibiotic group had a significantly higher proportion of bacteria resistant to ampicillin (p <0.01) and first-generation cephalosporin (p = 0.01) than the non-antibiotic group. In conclusion, there was a difference in bacterial flora in the sphenoid sinus mucosal samples collected before and after intraoperative antibiotic administration.

Snowfall reduces the risk of chronic subdural hematoma onset: Analysis of an administrative database in Japan.

BACKGROUND: Chronic subdural hematoma (CSDH) is a frequently observed disease in neurosurgical practice. Although first snowfall has been considered to increase the onset of CSDH, few reports have assessed the relationship between snowfall and CSDH. In this study, we aimed to investigate the relationship between CSDH onset and first snowfall events. METHODS: This retrospective study was based on the Japanese Diagnosis Procedure Combination inpatient database from July 1, 2010, to March 31, 2016, and on the global observation of snowfall events in Japan obtained from the Japan Meteorological Agency. We used a binomial approximation to evaluate the average number of CSDH patients after first snowfall events compared with that before first snowfall events. We calculated the odds ratio of CSDH onset on the first snowfall. RESULTS: We identified 8526 CSDH patients from the database. A total of 5573 (65.4%) were observed before first snowfall events, and 2953 (34.6%) after first snowfall events. The first snowfall of winter was significantly associated with a reduction in the occurrence of CSDH (odds ratio, 0.53; 95% confidence interval; 0.51-0.55). In subgroup analysis, only the first snowfall in October was not associated with reduction in the occurrence of CSDH. CONCLUSION: First snowfall events did not affect the onset of CSDH in Japan.

Optimization of immunohistochemical detection of rat ESR2 proteins with well-validated monoclonal antibody PPZ0506.

Although there are few well-validated antibodies against ESR2 proteins, a recent validation assessment identified a specific monoclonal antibody against human ESR2 proteins (PPZ0506). Furthermore, our previous study confirmed its cross-reactivity and specificity against rodent ESR2 proteins, enabling the determination of true ESR2 distribution profiles in rodents. Therefore, we aimed to determine optimal conditions for ESR2 detection by PPZ0506 immunostaining and analyze ESR2 distribution in rats. We evaluated several staining conditions using paraffin-embedded and frozen ovary sections. Immunohistochemical staining with PPZ0506 antibody required strong antigen retrieval and appropriate antibody dilution. Subsequent immunohistochemical analysis in multiple tissues under optimized conditions revealed that rat ESR2 proteins are expressed in a more localized manner than previously assumed. Our results suggest that previous immunohistochemical studies using inadequately validated antibodies against ESR2 proteins overestimated their distribution profiles. We expect that optimized immunohistochemical detection with PPZ0506 antibody can help researchers solve several conflicting problems in ESR2 research.