Keywords
cerebrum - confocal Imaging - Hoechst stain - z-stack
Introduction
The advancement of confocal microscopy in neurobiology, which is able to present cellular structures and processes from other tissues in the brain, is that it can put everything with high resolution. This is evidenced in the advanced application to the thicker brain tissues such as the cerebrum, which pose great challenges in light penetration and resolution at greater depths. The rat cerebrum is several millimeters thick that seriously presents complications in imaging; special methods are thus required to obtain a detailed observation of cell behavior, for example, in cell division. Knowledge of cell division in the cerebrum is important to understand neurogenesis, the mechanisms of brain development, and responses of the brain to injury and disease.
Hoechst stain is a common fluorescent dye that binds to DNA and useful in viewing nuclei, identifying cells undergoing mitosis. Combined with confocal microscopy, Hoechst staining can be used to visualize nuclear changes occurring in association with cell division in situ, providing critical insight into the dynamic behaviors of cells within the brain. However, for thick tissues like the cerebrum, great caution is required for optimization in the use of imaging parameters and at times even advanced techniques to enhance light penetration and reduce scattering.[1]
This staining assessed for the feasibility of confocal microscopy in observing cell division within thick rat cerebrum stained with Hoechst dye, using optimization of imaging conditions and employment of z-stack imaging to surmount the intrinsic problems arising from thick tissue imaging and provide further insight into cellular processes inside the cerebrum. These findings will add to the scaffolding of a better understanding of neurogenesis, and open new avenues for research on brain development and pathology.
Materials and Methods
Ethical Approval
All animal experimental procedures were performed with the approval of the Institute of Animal Ethics Committee and in accordance with guidelines for the care and use of laboratory animals.
Animal Housing and Rearing
Male Wistar albino rat (Rattus norvegicus) of 3 to 4 months old were individually housed in a temperature-controlled environment at 25°C. The animals received standard laboratory chow and water ad libitum, with hygiene as possible throughout the experiment.
Anesthesia
Intraperitoneal injection of ketamine hydrochloride (dose: 80 mg/kg body weight), along with Xylazine (dose: 8 mg/kg body weight) was utilized to euthanize the rats during the surgical processes.
Tissue Preparation and Extraction
Immediately after collection, rat brain was chopped into 2 mm thick slices, samples were washed with phosphate buffered saline and transferred into Dulbecco's Modified Eagle's Medium (DMEM) to keep the viability of the tissues for further staining.
Hoechst Staining
Stock solution preparation: for Hoechst stock solution, 100 mg of Hoechst dye was dissolved in 10 mL deionized water to make a stock solution of 10 mg/mL.
Preparation of staining solution: the stock solution (1 µL) was diluted in DMEM (10 µL) for making the working solution.
Staining procedure: Hoechst staining solution was directly placed to the prepared tissue samples and incubated for 5 to 15 minutes at 35°C in the dark to avoid photobleaching. After incubation, the staining solution was removed and washed three times with PBS to remove excess dye.
Imaging: stained tissues were imaged by a confocal laser scanning microscope (Leica SP8, Germany). Nuclear staining was observed with Hoechst dye ([Fig. 1]).
Fig. 1 Schematic representation of the methodology for sectioning the brain in a live media. The illustration depicts the step-by-step process, including removal of brain, sectioning of brain, and maintaining the tissue viability throughout the procedure by putting brain in live media. Key components such as temperature control, oxygenation, and media composition are highlighted to ensure optimal conditions for live brain sections.
Results
3D Confocal Imaging of a Thick Rat Cerebrum
Here, we demonstrate confocal microscopy imaging of nuclei in a thick section of rat cerebrum stained with Hoechst 33342 ([Fig. 2]). Hoechst staining labels the nuclei clearly and consistently to identify the cell bodies in the thick section of the brain. The fluorescence signal of the Hoechst dye was detected at 461 nm (emission wavelength), with excitation provided by a ultraviolet laser tuned at 350 to 365 nm. The nuclei showed strong fluorescence that assisted in clearly delineating nuclear boundaries deep inside the tissue. Considering the depth of a tissue section, imaging of a z-stack was performed to collect serial optical sections across the whole depth. Subsequent stacks were then combined to reconstruct a three-dimensional (3D) representation of tissue and show the spatial organization and density of nuclei within the whole sample ([Fig. 2]).
Fig. 2 Images (A–C) show detailed structural features of brain sections and cut section of the brain showing the size and thickness of brain, while (D) presents a 2D visualization of a single brain section, providing a flattened perspective. Images (E–G) depict 3D reconstructions of two brain sections, with (f) illustrating color-coded thickness variations to highlight structural differentiation.
The resultant 3D image reconstruction showed that indeed the overall distribution of nuclei across the cerebrum was rather uniform, with a slight increase in density within the cortical regions compared with deeper white matter. These were even more pronounced in cortical regions with the 3D rendering, which also allowed for the observation of nuclear clustering and alignment along certain anatomical structures. Expected in the case of thick tissue imaging, some fluorescence signal attenuation with increasing depth was due to the limitations of confocal microscopy. However, while nuclei were still distinguishable up to about 1.5 mm deep with some loss of intensity, a significant decay of signal intensity beyond that point suffered resolution of nuclear features. This attenuation underlines the challenges of thick section imaging but at the same time points to the robustness of Hoechst stain for consistent nuclear labeling even in deeper tissue layers.
Discussion
The current study succeeded in confocal imaging and analysis of cellular architecture in a thick sectioned rat cerebrum with accurate nuclear labeling by Hoechst 33342 staining and further 3D reconstruction from z-stacks, which gave the complete overview of nuclear distribution through this large volume of tissue. One of the major disadvantages with confocal microscopy is that high-resolution imaging is restricted to thin sections of tissues. In our study, although signal attenuation was noted beyond 1.5 mm, nuclei were still visible even in depth, thus allowing a demonstration that with excellent staining and imaging parameters, effective good imaging depth may be achieved. Indeed, this finding agrees with several previous reports showing the possibility of deeper tissue imaging when using confocal microscopy, although there are several trade-offs involved with regard to resolution and signal intensity.[2]
[3] In this way, the 3D reconstruction of the cerebrum offered a detailed spatial map of nuclear organization that would have been quite difficult to discern with only 2D sections. Increased nuclear density in cortical regions, as seen in this work, is consistent with the established view regarding cortical layering and cellular heterogeneity present in the rat brain.[4] Therefore, such 3D imaging techniques have a great value in neuroanatomical studies because they allow more accurate modeling of the structures of the human brain and thus could lead to a better understanding of functional connectivity and cortical organization. Imaging thick brain sections by confocal microscopy opens new dimensions for investigations into the structural and cellular changes occurring in a variety of neurological diseases. Thus, this technique can be applied in brains with neurodegenerative conditions where subtle changes in cell density and distribution may well precede more obvious pathological signs of the disease.[5] In principle, therefore, a combination of confocal microscopy with two-photon microscopy or even other imaging modalities may further improve depth and resolution of images obtained with brain imaging and thus reveal more about the detailed features of brain architecture.[1] While the present approach allowed the imaging of nuclei throughout most of the 2 mm thick tissue, there are some limitations that need to be considered. More importantly, a decrease in the signal intensity at greater depths reflects an intrinsic limitation of using single-photon confocal microscopy for the imaging of thick tissues. Techniques of clearing, such as CLARITY or iDISCO, which make the tissues transparent and allow for much greater depth of imaging, could be used in future experiments.[6]
[7] In addition, it would also be very useful to incorporate an adaptive optics approach to confocal microscopy to correct for aberrations introduced by optical properties of tissues and improve image quality at deeper levels.[8]
Conclusion
This study thus demonstrates the feasibility and utility of confocal microscopy to perform three-dimensional imaging of thick brain tissue sections and thus for investigating nuclear architecture in rat cerebrum. Although signal attenuation remains a challenge in imaging thick tissue sections, judicious staining, imaging, and reconstruction techniques provide a formidable tool for neuroanatomical studies. Such techniques, when further refined, are likely to be widely employed in basic research and clinical investigations of the brain.
