A procedure in mice to obtain intact pituitary-infundibulum-hypothalamus preparations: a method to evaluate the reconstruction of hypothalamohypophyseal system.

PURPOSE: The histopathological study of brain tissue is a common method in neuroscience. However, efficient procedures to preserve the intact hypothalamic-pituitary brain specimens are not available in mice for histopathological study. METHOD: We describe a detailed procedure for obtaining mouse brain with pituitary-hypothalamus continuity. Unlike the traditional methods, we collect the brain via a ventral approach. We cut the intraoccipital synchondrosis, transection the endocranium of pituitary, broke the spheno-occipital synchondrosis, expose the posterior edge of pituitary, separate the trigeminal nerve, then the intact pituitary gland was preserved. RESULT: We report an more effective and practical method to obtain continuous hypothalamus -pituitary preparations based on the preserve of leptomeninges. COMPARED WITH THE EXISTING METHODS: Our procedure effectively protects the integrity of the fragile infundibulum preventing the pituitary from separating from the hypothalamus. This procedure is more convenient and efficient. CONCLUSION: We present a convenient and practical procedure to obtain intact hypothalamic-pituitary brain specimens for subsequent histopathological evaluation in mice.

Adult neurogenesis of the median eminence contributes to structural reconstruction and recovery of body fluid metabolism in hypothalamic self-repair after pituitary stalk lesion.

Body fluid homeostasis is critical to survival. The integrity of the hypothalamo-neurohypophysial system (HNS) is an important basis of the precise regulation of body fluid metabolism and arginine vasopressin (AVP) hormone release. Clinically, some patients with central diabetes insipidus (CDI) due to HNS lesions can experience recovery compensation of body fluid metabolism. However, whether the hypothalamus has the potential for structural plasticity and self-repair under pathological conditions remains unclear. Here, we report the repair and reconstruction of a new neurohypophysis-like structure in the hypothalamic median eminence (ME) after pituitary stalk electrical lesion (PEL). We show that activated and proliferating adult neural progenitor cells differentiate into new mature neurons, which then integrate with remodeled AVP fibers to reconstruct the local AVP hormone release neural circuit in the ME after PEL. We found that the transcription factor of NK2 homeobox 1 (NKX2.1) and the sonic hedgehog signaling pathway, mediated by NKX2.1, are the key regulators of adult hypothalamic neurogenesis. Taken together, our study provides evidence that adult ME neurogenesis is involved in the structural reconstruction of the AVP release circuit and eventually restores body fluid metabolic homeostasis during hypothalamic self-repair.

Transcriptomic Analysis Reveals that Activating Transcription Factor 3/c-Jun/Lgals3 Axis Is Associated with Central Diabetes Insipidus after Hypothalamic Injury.

BACKGROUND: Hypothalamic injury causes several complicated neuroendocrine-associated disorders, such as water-electrolyte imbalance, obesity, and hypopituitarism. Among these, central diabetes insipidus (CDI), characterized by polyuria, polydipsia, low urine specific gravity, and deficiency of arginine vasopressin contents, is a typical complication after hypothalamic injury. METHODS: CDI was induced by hypothalamic pituitary stalk injury in male animals. Behavioral parameters and blood sample were collected to evaluate the characteristics of body fluid metabolism imbalance. The brains were harvested for high-throughput RNA sequencing and immunostaining to identify pathophysiological changes in corresponding hypothalamic nuclei. RESULTS: Based on transcriptomic analysis, we demonstrated the upregulation of the activating transcription factor 3 (Atf3)/c-Jun axis and identified Lgals3, a microglial activation-related gene, as the most significant target gene in response to the body fluid imbalance in CDI. Furthermore, we found that the microglia possessed elevated phagocytic ability, which could promote the elimination of arginine vasopressin neurons after hypothalamic injury. CONCLUSION: Our findings suggested that the Atf3/c-Jun/Lgals3 axis was associated with the microglial activation, and might participate in the loss of functional arginine vasopressin neurons in CDI after hypothalamic injury.

Growth hormone promotes the reconstruction of injured axons in the hypothalamo-neurohypophyseal system.

JOURNAL/nrgr/04.03/01300535-202410000-00026/figure1/v/2024-02-06T055622Z/r/image-tiff Previous studies have shown that growth hormone can regulate hypothalamic energy metabolism, stress, and hormone release. Therefore, growth hormone has great potential for treating hypothalamic injury. In this study, we established a specific hypothalamic axon injury model by inducing hypothalamic pituitary stalk electric lesions in male mice. We then treated mice by intraperitoneal administration of growth hormone. Our results showed that growth hormone increased the expression of insulin-like growth factor 1 and its receptors, and promoted the survival of hypothalamic neurons, axonal regeneration, and vascular reconstruction from the median eminence through the posterior pituitary. Altogether, this alleviated hypothalamic injury-caused central diabetes insipidus and anxiety. These results suggest that growth hormone can promote axonal reconstruction after hypothalamic injury by regulating the growth hormone-insulin-like growth factor 1 axis.

An Efficient Method for the Isolation and Cultivation of Hypothalamic Neural Stem/Progenitor Cells From Mouse Embryos.

The hypothalamus is the key region that regulates the neuroendocrine system as well as instinct behaviors, and hypothalamic dysfunction causes refractory clinical problems. Recent studies have indicated that neural stem/progenitor cell (NSPC) in the hypothalamus play a crucial role in hypothalamic function. However, specific hypothalamic NSPC culture methods have not been established, especially not detailed or efficient surgical procedures. The present study presented a convenient, detailed and efficient method for the isolation and cultivation of hypothalamic NSPCs from embryonic day 12.5 mice. The procedure includes embryo acquisition, brain microdissection to quickly obtain hypothalamic tissue and hypothalamic NSPC culture. Hypothalamic NSPCs can be quickly harvested and grow well in both neurosphere and adherent cultures through this method. Additionally, we confirmed the cell origin and evaluated the proliferation and differentiation properties of cultured cells. In conclusion, we present a convenient and practical method for the isolation and cultivation of hypothalamic NSPCs that can be used in extensive hypothalamic studies.

A retaining sphenoid and dura procedure in the rat to obtain intact pituitary-infundibulum-hypothalamus preparations.

BACKGROUND: The histopathological study of brain tissue is a conventional method in neuroscience. However, procedures specifically developed to recover intact hypothalamic-pituitary brain specimens, are not available. NEW METHOD: We describe a detailed protocol for obtaining intact rat brain with pituitary-hypothalamus continuity through an intact infundibulum. The brain is collected via a ventral approach through removing the skull base. Membranous structures surrounding the hypothalamus-pituitary system can be preserved, including vasculature. RESULTS: We report a retaining sphenoid and dura technique to obtain intact hypothalamic-pituitary brain preparations, and we confirm the practicability of this method. By combination of this technique with histological analysis or 3D brain tissue clearing and imaging methods, the functional morphology structure of the hypothalamus-pituitary can be further explored. COMPARISON WITH EXISTING METHOD: The current procedure is limited in showing the connection between the hypothalamus and the pituitary. Our procedure effectively protects the integrity of the fragile infundibulum and thus prevents the pituitary from separating from the hypothalamus. CONCLUSIONS: We present a convenient and practical approach to obtain intact hypothalamus-pituitary brain specimens for subsequent histopathological evaluation.

[JNK/c-Jun signaling pathway mediates arginine vasopressin neuron regeneration by promoting cytoskeleton reconstruction in rats with electrical lesions of the pituitary stalk].

OBJECTIVE: To investigate the mechanism by which doublecortin promotes the recovery of cytoskeleton in arginine vasopressin (AVP) neurons in rats with electrical lesions of the pituitary stalk (PEL). METHODS: Thirty-two SD rats were randomized into PEL group with electrical lesions of the pituitary stalk through the floor of the skull base (n=25) and sham operation group (n=7), and the daily water consumption (DWC), daily urine volume (DUV) and urine specific gravity (USG) of the rats were recorded. Four rats on day 1 and 7 rats on each of days 3, 7 and 14 after PEL as well as the sham-operated rats were sacrificed for detection of the expressions of ß-Tubulin (Tuj1), doublecortin and caspase- 3 in the AVP neurons of the supraoptic nucleus using immunofluorescence assay and Western blotting. RESULTS: After PEL, the rats exhibited a typical triphasic pattern of diabetes insipidus, with the postoperative days 1-2 as the phase one, days 3-5 as the phase two, and days 6-14 as the phase three. Immunofluorescent results indicated the repair of the AVP neurons evidenced by significantly increased doublecortin expressions in the AVP neurons following PEL; similarly, the expression of Tuj1 also increased progressively after PEL, reaching the peak level on day 7 after PEL. The apoptotic rates of the AVP neurons exhibited a reverse pattern of variation, peaking on postoperative day 3 followed by progressive reduction till day 14. Western blotting showed that the expressions of c-Jun and p-c-Jun were up-regulated significantly on day 3 (P < 0.05) and 7 (P < 0.01) after PEL, while an upregulated p-JNK expression was detected only on day 3 (P < 0.05), as was consistent with the time-courses of neuronal recovery and apoptosis after PEL. CONCLUSIONS: JNK/c-Jun pathway is activated after PEL to induce apoptosis of AVP neurons in the acute phase and to promote the repair of neuronal cytoskeleton by up-regulation of doublecortin and Tuj1 expressions.

Endoplasmic reticulum stress induces apoptosis of arginine vasopressin neurons in central diabetes insipidus via PI3K/Akt pathway.

AIMS: Central diabetes insipidus (CDI), a typical complication caused by pituitary stalk injury, often occurs after surgery, trauma, or tumor compression around hypothalamic structures such as the pituitary stalk and optic chiasma. CDI is linked to decreased arginine vasopressin (AVP) neurons in the hypothalamic supraoptic nucleus and paraventricular nucleus, along with a deficit in circulating AVP and oxytocin. However, little has been elucidated about the changes in AVP neurons in CDI. Hence, our study was designed to understand the role of several pathophysiologic changes such as endoplasmic reticulum (ER) stress and apoptosis of AVP neurons in CDI. METHODS: In a novel pituitary stalk electric lesion (PEL) model to mimic CDI, immunofluorescence and immunoblotting were used to understand the underlying regulatory mechanisms. RESULTS: We reported that in CDI condition, generated by PEL, ER stress induced apoptosis of AVP neurons via activation of the PI3K/Akt and ERK pathways. Furthermore, application of N-acetylcysteine protected hypothalamic AVP neurons from ER stress-induced apoptosis through blocking the PI3K/Akt and ERK pathways. CONCLUSION: Our findings showed that AVP neurons underwent apoptosis induced by ER stress, and ER stress might play a vital role in CDI condition through the PI3K/Akt and ERK pathways.