Volumetric parcellation methodology of the human hypothalamus in neuroimaging: Normative data and sex differences

There is increasing evidence regarding the importance of the hypothalamus for understanding sex differences in relation to neurological, psychiatric, endocrine and sleep disorders. Although different in histology, physiology, connections and function, multiple hypothalamic nuclei subserve non-voluntary functions and are nodal points for the purpose of maintaining homeostasis of the organism. Thus, given the critical importance of hypothalamic nuclei and their key multiple roles in regulating basic functions, it is important to develop the ability to conduct in vivo human studies of anatomic structure, volume, connectivity, and function of hypothalamic regions represented at the level of its nuclei. The goals of the present study were to develop a novel method of semi-automated volumetric parcellation for the human hypothalamus that could be used to investigate clinical conditions using MRI and to demonstrate its applicability. The proposed new method subdivides the hypothalamus into five parcels based on visible anatomic landmarks associated with specific nuclear groupings and was confirmed using two ex vivo hypothalami that were imaged in a 7 T (7 T) scanner and processed histologically. Imaging results were compared with histology from the same brain. Further, the method was applied to 44 healthy adults (26 men; 18 women, comparable on age, handedness, ethnicity, SES) to derive normative volumes and assess sex differences in hypothalamic regions using 1.5 T MRI. Men compared to women had a significantly larger total hypothalamus, relative to cerebrum size, similar for both hemispheres, a difference that was primarily driven by the tuberal region, with the sex effect size being largest in the superior tuberal region and, to a lesser extent, inferior tuberal region. Given the critical role of hypothalamic nuclei in multiple chronic diseases and the importance of sex differences, we argue that the use of the novel methodology presented here will allow for critical investigations of these disorders and further delineation of potential treatments, particularly sex-specific approaches to gene and drug discoveries that involve hypothalamic nuclei

Development of Microstructural Segmentation and 3D Reconstruction Method Using Serial Section of Tissue: 3D Educational Model of Human Hypothalamus

학위논문(석사)--서울대학교 대학원 :의과대학 의학과,2019. 8. 황영일최형진.INTRODUCTION: The 3D reconstruction technique of tissue staining images is very valuable in that it visualizes the microstructure information that Magnetic resonance imaging (MRI) and Computed tomography (CT) data cannot provide and is widely used for pathological diagnosis. Organizational 3D reconstruction needs the latest devices and software for each phase. However, in reality, it is not easy to equip all of the most brand-new equipment, and software, so the existing research has been done by only a limited number of people. For this reason, in this study, we tried to develop a 3D reconstruction method of the organization by using available laboratory equipment and highly accessible software. The human hypothalamus is relatively small compared to other brain structures, but it is the backbone of homeostasis regulation and an important structure directly linked to survival. It consists of more than 13 nuclei and microstructures, and many attempts have been made to identify them. However, most reported histology images were based on the 2D map, that cause researchers are experiencing difficulties in understanding spatial structure perception. In addition, most of the currently reported hypothalamic 3D maps are based on MRI data. This DICOM based medical image has the disadvantage that it is difficult to understand the detailed microstructure of the nucleus. In order to overcome these drawbacks, this study aims to develop a detailed 3D model of the human hypothalamus by using easily accessible devices and software. Since the 3D map of the human hypothalamus has not been reported so far, we have developed a method that allows a wider range of researchers to perform the 3D reconstruction of the tissue, which had previously been done by a limited number of people. We also tried to make the model created by the researchers easily accessible to the field. Methods: Nissl staining of human brain hypothalamus obtained by autopsy was converted to a digital image using a tissue slide scanner. The whole slide image was converted by using image processing software to adjust the resolution and extension. After that, segmentation of the hypothalamic microstructure was performed in the Adobe Photoshop software, and the missing slide images were prepared by manual interpolation. All the structure segmented images were transformed into black and white to produce a mask suitable for 3D reconstruction, and they were classified by structure. Then, the whole image was subjected to bit number correction and extension conversion suitable for 3D reconstruction using ImageJ software. Then 3D reconstruction software reconstructs the segmented structures into three dimensions and attempts 3D rendering. After transforming them into STL extensions, we tried to edit them using MeshMixer software. Through this process, 3D map was created with WebGL, and the 3D map education model of the human hypothesis was created. Results: A total of 100 staining images were obtained by Nissl staining using human brain hypothalamus. To make our results more clearly, hypothalamic 2D maps obtained in this study were compared with Allen atlas. A total of 23 segmentations were carried out including hypothalamic surrounding structures and nucleus distribution patterns. A total of 11 excluded slides were supplemented by manual interpolation. The hypothalamus 2D map was used to reconstruct the human hypothalamus as a 3D reconstructed volume model and a 3D reconstructed surface model. The 3D reconstruction surface model was obtained by using MeshMixer to complement the smoothing and the outlier point of each structure. Then, I created a hypothalamus 3D reconstruction education model using WebGL service to make possible for anyone to easily access and learn without the constraint of time and space. Discussion: In this study, I developed a method for producing 2D map and 3D reconstructed images of Nissl stained using hypothalamus tissue. This is the first 3D reconstruction model based on the hypothalamus, which is meant to help other researchers and medical personnel in education and research. Previous studies have shown that the spacing of the slices of the hypothalamus tissue was not constant, but this study succeeded in acquiring the results of the staining of the hypothalamus tissue at 100 ㎛ intervals as the basic data for 3D reconstruction. Many other types of missing images were found due to the lack of consideration of various variables that occurred during the reconstruction process. The anatomical structure and various parameters were considered and corrected for more satisfactory results. In addition, existing image-based software provides automatic segmentation function considering only the distinctive features of shaded images, so it is very inappropriate to classify subtle clustering patterns such as nucleus and structures in the human hypothalamus. It is significant that the progress process is segmented, and the separate software suitable for each process is applied, and the process of working with them is compatible with each other. Most software is free, low cost and easy to learn and use, so it provides a way to easily create an organization 3D image without expensive software or equipment. The existing hypothalamus training data were mostly 2D illustrations, but the 3D reconstructed images produced in this study are easy to grasp the positional relationship of structures more space. In particular, since the hypothalamus does not contain data showing nuclear reconstruction as a 3D reconstructed image, the educational model of this study will be of great help to many hypothalamus researchers. And the 3D WebGL education model has pedagogical value because it enables free access and access through users personal device, enabling ubiquitous learning that is not restricted by time and space. Conclusions: Through this study, I have established a method for producing 2D map and 3D reconstruction using human hypothalamus. Through the 3D reconstruction image and the education model, the positional relation of the human hypothalamus can be recognized by spatial perception. This result is pedagogically worthy because it can be used as U-learning material to help researchers self-directed learning by opening it to open source WebGL for easy use by anyone.서론: 조직 염색 영상의 3차원 재구성 기술은 MRI와 CT가 제공하지 못하는 미세구조를 시각화 하여 3차원 조직학에 활용된다는 점에서 가치가 있다. 조직 3차원 재구성에는 각 단계에 적합한 기기와 소프트웨어가 사용된다. 그러나 고가의 기기와 소프트웨어를 전부 갖추기란 쉽지 않으므로 기존의 연구는 제한적인 소수에 의해 이루어져 왔다. 사람 시상하부는 다른 뇌 구조물에 비해 상대적으로 크기는 작지만 항상성 조절의 중추이며 생존과 직결된 신체 활동을 조절하는 중요한 기관이다. 기존 시상하부에 대한 시각적 연구는 조직학 영상 기반의 2차원 지도 중심으로 이루어져 구조를 공간지각적으로 이해하는 데 많은 어려움이 있었다. 또한 현재 발표된 대부분의 사람 시상하부 3차원 지도는 MRI 데이터를 기반으로 작성되어 있어 신경핵 단위의 미세구조 정보를 충분히 전달하지 못한다. 따라서 본 연구에서는 두가지 목표를 달성하고자 하였다. 첫째로, 접근성이 좋은 장비와 소프트웨어를 활용하여 보다 넓은 범위의 연구자들이 수행할 수 있는 조직의 3차원 재구성 방법을 개발하고자 하였다. 둘째로, 위의 과정을 통해 확립한 방법을 시상하부에 적용하여 해당 분야 연구자들이 학습 및 교육에 활용할 수 있는 3차원 모델을 제작하고자 하였다. 연구 대상 및 방법: 사람 시상하부 전체와 시각로가 포함된 조직을 대상으로 조직 3차원 재구성을 시도하였다. 염색된 각 조직절편을 슬라이드 스캐너를 이용해 디지털 영상으로 변환하였고 ZEN을 사용하여 해상도 조절과 확장자 변환을 시행하였다. 전체 영상을 Adobe Photoshop을 이용하여 시각로와 미세혈관, 안쪽후각겉질의 외곽선을 기준으로 정합 하였다. 이 후 미세조직 구역화, 소실된 슬라이드 영상의 수동 보간법 적용, 전체 구역화 영상의 흑백 변환, 마스크 제작, 구조물 별 분류를 시행하였다. 또한 전체 영상을 ImageJ를 사용하여 bit수 교정 및 확장자 변환을 수행하였다. 이 후 MEDIP에서 구역화 한 구조물 영상의 3차원 재구성, STL 확장자 변환 및 내보내기를 시행하였다. 이 후 MeshMixer를 사용하여 표면의 요철과 이상점을 교정한 뒤 webGL 교육모델로 제작하였다. 이렇게 수립된 protocol을 사람 시상하부에 적용하여 내부 신경핵과 미세구조를 3차원으로 재구성하였다. 결과: Zen, Adobe Photoshop, ImageJ를 사용하여 조직 염색 영상, 시각로 2차원 지도, 색면 레이어, 패스영역 레이어, 흑백변환 영상, 흑백 반전 영상, Raw data mask를 생성하였다. 이 후 MEDIP과 Meshmixer 를 사용하여 전체조직과 시각로 부피모델, 표면모델, 교육모델을 생성하였다. 이를 사람 시상하부 미세구조의 시각화에 적용하여 조직 염색 영상, 2차원 지도, 미세구조와 신경핵 구역화 색면 레이어, 패스영역 레이어, 흑백 변환 영상, 흑백 반전 영상, 3차원 Raw data mask, 3차원 재구성 부피 모델, 표면 모델, 3차원 교육모델을 생성하였다. 고찰: 본 연구에서는 Zen, Adobe Photoshop, ImageJ, MEDIP, Meshmixer로 이어지는 조직의 3차원 재구성 제작 protocol을 개발하였다. 본 연구는 기존의 3차원 재구성 방법론과 비교했을 때 외곽선이 뚜렷하지 않은 구조물의 영상 위에 수동으로 구역화를 수행했다는 점에서 차별성이 있다. 또한 기존의 조직 3차원 재구성 방법론과 비교했을 때 각 단계에 사용되는 고가의 소프트웨어와 기기를 여러 개의 접근성이 높은 소프트웨어로 분할 및 적용했다는 점에서 기존 연구와 다르다. 본 연구의 2차원 지도는 Allen atlas가 제공하는 사람 뇌 2차원 지도와 비교했을 때 보다 촘촘한 100 ㎛의 영상을 일정한 간격으로 획득했다는 점에서 차별성이 있다. 또한 MRI 기반 3차원 재구성 모델이 제공하지 못하는 여러 미세구조와 신경핵을 시각화 했다는 점에서 가치가 있다. 기존의 2차원 신경해부학 교육자료와 비교했을 때 본 연구에서 제작한 3차원 교육모델은 구조물들의 위치관계를 공간지각적으로 보다 쉽게 파악할 수 있다는 장점이 있다. 또한 본 연구의 결과물은 기존의 3차원 신경해부학 교육자료와 달리 실험을 통해 얻은 실물 자료를 기반으로 제작되었다는 점에서 가치가 있다. 결론: 본 연구에서는 사람 시상하부 조직을 매개로 조직의 3차원 재구성 protocol을 확립하였다. 이를 통해 사람 시상하부 내부의 미세구조와 신경핵의 위치관계를 공간지각적으로 파악할 수 있는 부피모델과 표면모델을 생성할 수 있었다. 이 결과물들은 의료인 교육에 적합한 교육모델로 변환하여 공개 자료로 제공되었다.초 록 ……………………………………………………………ⅰ 목 차 …………………………………………………………… ⅳ 표 목록 ……………………………………………………… v 그림 목록…………………………………………………… vi 서 론…………………………………………………………… 9 본 론 …………………………………………………………… 15 Chapter 1. 조직의 3차원 재구성 제작 protocol 개발...... 15 Chapter 2. 사람 시상하부 조직의 3차원 재구성.............. 52 결 론…………………………………………………………… 95 참고문헌…………………………………………………………96 Abstract………………………………………………………101Maste

Molecular studies of hypothalamic food intake regulating systems and central myelination in two anorectic mouse models

The aim of this thesis is to increase the understanding of the hypothalamic mechanisms that are vital for food intake regulation, in particular as it relates to anorexia, and the molecular mechanisms underlying myelination and nodal/paranodal domain organization. The majority of the thesis concerns the anorectic phenotype of two mouse models for which we have found both great similarities and differences pointing to different mechanisms generating similar symptoms and neuropeptidergic alterations. This thesis starts with immunohistological and electron microscopic investigations of the regulatory effect of the GPI-anchored cell adhesion molecule Contactin-1 in myelination in the central nervous system (CNS) and functional compartmentalization of myelinated nerves (Paper I). With the help of the Cntn1-KO mouse and a Cntn1-KO cross expressing green fluorescent protein in myelin, our analysis revealed several novel functions of Contactin-1 in CNS myelination. These include the demonstration of Contactin-1 expression in oligodendrocytes in vivo and its role in regulating neuron-glia interactions required for myelin membrane extension and myelination. Further, we found that Contactin-1 is essential for the domain organization of myelinated nerves by organizing the attachment of the terminal myelin loops to the axon membrane at the paranode. Contactin-1 is thus a key molecule for forming functional fast propagating high conduction velocity myelinated nerves in the CNS. We continue to explore the roles of Contactin-1 in the CNS by investigating the anorectic and hypothalamic phenotype of the Cntn1-KO in comparison with the anorectic anx/anx mouse (Paper II and IV). In these two studies involving immunohistochemistry and in situ hybridization techniques, we show similarities between the two models in the deviation from the wild type expression levels and location of hypothalamic neuropeptides (NPY, AgRP, α-MSH/POMC and MCH) important for food intake regulation (Paper II). However, further analysis revealed apparent differences with regard to the expression of astroglial and microglial markers in the hypothalamic system, as well as in the hippocampus (Paper IV). A significant upregulation of markers of astroglial and of microglial activation (previously published) was found in the anx/anx hypothalamus, indicating an inflammatory reaction. In contrast, the Cntn1-KO mouse displays no such glial responses in the hypothalamus. We did however detect increased expression of the microglia marker in the hippocampal dentate gyrus of the Cntn1-KO mouse, which we did not see in the anx/anx mouse. Based on previous findings associating the anx/anx mouse with a mitochondrial dysfunction, we explored the possibility of a reduced metabolic rate of hypothalamic neurons (Paper III). Enzymatic assays, ex vivo autoradiography and Western Blot of the anx/anx hypothalamus revealed reduced glucose uptake, reduced cellular metabolic rate both in basal and ischemic conditions and reduced ATP-turnover. The ratio of the metabolic master regulator, AMPK-P/AMPK, was reduced in the anorectic anx/anx hypothalamus. Taken together this is indicative of a hypometabolic state in the hypothalamus of the anx/anx mouse resembling what is seen during hibernation. The two anorectic mouse models have many similarities and many differences making them valuable to further understand the food intake regulating systems. By elucidating molecular pathways the data in this thesis may in the future yield improved understanding of disorders such as Anorexia Nervosa and Multiple Sclerosis

Novel aspects of insulin resistance: focus on the brain. Studies using positron emission tomography

We are currently facing a global epidemic of obesity, which poses a great challenge for the global health. Insulin resistance is the common soil that links obesity and type 2 diabetes. Whereas most lifestyle interventions fail, bariatric surgery has been a powerful weapon in the battle against obesity. Preclinical data have shown that the brain may directly control the determinants of glucose tolerance, namely insulin sensitivity and insulin secretion. We used positron emission tomography, to study whether brain metabolism assessed as brain glucose and/or brain fatty acid uptake is related to the endogenous glucose production (EGP), and ß-cell function. Moreover, we addressed whether there are differences in brain fatty acid uptake between morbidly obese and lean individuals as well as the effect of significant weight loss induced by bariatric surgery. Finally, we investigated whether brain substrate handling predicted any metabolic outcome at follow-up. We found that in morbidly obese subjects brain glucose uptake (BGU) correlated positively with EGP, and that this association remained significant also six months after surgery. On the contrary, there was no such association in the lean subjects. In 52 non-diabetic subjects to whom ß-cell modeling was performed, insulin-stimulated BGU correlated also with the basal insulin secretion, total insulin output and potentiation of glucose-stimulated insulin secretion. Contrastingly, in 15 patients with type 2 diabetes BGU and insulin secretion did not correlate, but there was a significant inverse correlation between BGU and potentiation. Cross-sectionally in 34 studied women, brain fatty acid uptake (BFAU) also correlated negatively with potentiation, and similar trends were seen both in non-diabetics and diabetics. Finally, we found that, unlike in lean individuals, BFAU is increased in morbidly obese subjects, and that six months after bariatric surgery, BFAU remained unchanged. Baseline BGU and baseline BFAU predicted worse glucose control at two-year follow-up after bariatric surgery. In conclusion, this thesis work shows that brain substrate handling differs in obese and lean individuals and that brain metabolism may be a direct way of controlling whole-body homeostasis in humans. Moreover, in two different datasets, increased brain substrate uptake at baseline predicted worse metabolic outcome after bariatric surgery

Sex steroid hormones-related structural plasticity in the human hypothalamus.

International audienceWe investigated the effects of an artificial menstrual cycle on brain structure and activity in young women using metabolic magnetic resonance imaging (MRI). We show that the activation of the hypothalamo-pituitary-gonadal axis during the pill-free interval of low-dose combined oral contraceptive use is associated with transient microstructural and metabolic changes in the female hypothalamus but not in the thalamus, a brain structure unrelated to reproductive control, as assessed by water diffusion and proton magnetic resonance spectra measurements. Our results provide neuroanatomical insights into the mechanism by which sex steroid hormones mediate their central effects and raise the intriguing possibility that specific regions of the neuroendocrine brain use ovarian cycle-dependent plasticity to control reproduction in humans. These MRI-based physiological studies may pave the way for the development of new diagnostic and treatment strategies in the central loss of reproductive competence in human syndromes, such as hypothalamic amenorrhea

Calidad de la respuesta estrogénica por influencia de las hormonas tiroideas

Tesis- Doctor en Medicina y Cirugía- Universidad Nacional de Córdoba. Facultad de Ciencias Médicas, 2019Introduction: The normal plasma estrogen values associated with the upper limit of TSH and normal or low T4L, act on the estrogen’s effectors while female reproductive age. This situation causes metabolic alterations that compromise ovarian function, decrease ovulatory capacity, previously adjusted to the subclinical hypothyroidism. Material and Method: 813 women were evaluated (between 21 and 41 years old), discriminated according to the values of TSH and FT4, in 3 groups: "without pathology, G-1"; "with pathology, G-2" and "undefined, G3". Appointments for menstrual disorders and reproductive desire, were relevant in “G-2” and “G-3”. The variables evaluated were: BMI and nutritional status; glucidic and lipid profile. The thyroid evaluation included: functional, immunological and morphological study. Finally, ovarian function: FSH - LH - Estradiol and Prolactin, hormonal colpocytology and gynecological ultrasound. Results: age, origin, age of menarche and initiation of sexual relations, shows parity between the groups. The metabolic results showed that in the "G-3", the percentage of overweight patients was intermediate between “G1” and “G-2”; the level of triglycerides was higher in “G-1” but lower than “G2”, and the insulin was slightly increased, but within normal values. The thyroid evaluation revealed significant differences of the "G-3" in relation to “G-1” and “G-2”. The ovarian profile revealed statistically significant differences for LH between “G-3” vs. “G-1” and “G-2”. The finding of ovarian alterations at ultrasounds showed the presence of micro polycystic ovaries with also significant differences. Conclusion: this work shows that the normalization of TSH and FT4 values restore normal ovarian function, jointly with the remission of gynecological alterations that constituted the initial reason for complain. The number of pregnancies achieved in the "indefinite group, G-3" (54 pregnancies, 79.4%) confirms the proposed hypothesis.Introducción: valores de estrógenos plasmáticos normales asociados a TSH en el límite superior y Tac normal o baja, afectan los electores estrogénicos durante la edad reproductiva femenina. Esta situación, ocasiona alteraciones metabólicas que comprometen la función ovárica, disminuyendo la capacidad ovulatoria, previa a configurar el cuadro de hipotiroidismo subclínico. Material y Método: fueron evaluadas 813 mujeres, entre 21 y 41 años discriminadas según los valores de TSH y T4L, en 3 grupos: "sin patología. G-1"; "con patología, G-2" e "indefinidas, G-3". Las consultas, por trastornos menstruales y deseo reproductivo, resultaron relevantes en G-2 y G-3. Las variables evaluadas fueron: IMC y estado nutricional; perfil glucidico y lipídico. La evaluación tiroidea incluyó: estudio funcional. inmunológico y morfológico. Por último, función ovárica: FSH — LH — Estradiol y Prolactina, colpocitología hormonal y ecografía ginecológica. Resultados: la edad, procedencia, edad de menarca e inicio de relaciones sexuales, muestra paridad entre los grupos. Los resultados metabólicos, mostraron que en el "grupo G-3", el porcentaje de pacientes con sobrepeso se encontraba en cifras intermedias entre G-1 y G-2; el nivel de triglicéridos era más elevado en G-1 pero inferior a G-2, y la insulina estaba levemente aumentada, pero dentro de valores normales. La valoración tiroidea, reveló diferencias significativas del "grupo G-3" con relación a G-1 y G-2. El perfil ovárico, reveló diferencias estadísticamente significativas para LH entre G-3 vs G-1 y G-2. El hallazgo de alteraciones ováricas ecografías mostró la presencia de ovarios micropoliquísticos con diferencias también significativas. Conclusión: el motivo de la presente investigación, demuestra que la normalización de los valores TSH y T4L restablecen el normal funcionamiento ovárico, y con él la remisión de las diversas alteraciones ginecológicas que constituyeron el motivo inicial de consulta. Entre los cuales el número de embarazos logrados en el "grupo indefinidas, G-3" (54 embarazos, 79,4%) fue una variable representativa que permitió objetivar la confirmación de la hipótesis planteada.2021-10-22Fil: Espinosa, Pedro Conrado. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas; Argentina