Primo Vascular System in the Subarachnoid Space of a Mouse Brain

Objective. Recently, a novel circulatory system, the primo vascular system (PVS), was found in the brain ventricles and in the central canal of the spinal cord of a rat. The aim of the current work is to detect the PVS along the transverse sinuses between the cerebrum and the cerebellum of a mouse brain. Materials and Methods. The PVS in the subarachnoid space was analyzed after staining with 4′,6-diamidino-2-phenylindole (DAPI) and phalloidin in order to identify the PVS. With confocal microscopy and polarization microscopy, the primo vessel underneath the sagittal sinus was examined. The primo nodes under the transversal sinuses were observed after peeling off the dura and pia maters of the brain. Results. The primo vessel underneath the superior sagittal sinus was observed and showed linear optical polarization, similarly to the rabbit and the rat cases. The primo nodes were observed under the left and the right transverse sinuses at distances of 3,763 μm and 5,967 μm. The average size was 155 μm × 248 μm. Conclusion. The observation of primo vessels was consistent with previous observations in rabbits and rats, and primo nodes under the transverse sinuses were observed for the first time in this work.


Introduction
The primo vascular system (PVS) was proposed by Kim as a third circulatory system that corresponded to and extended the acupuncture meridians [1]. In various parts of an animal's body, the PVS was confirmed [2], especially in blood vessels [3], the heart [4], lymph vessels [5,6], and on the surfaces of internal organs [7] of mice, rats, and rabbits.
The PVS in the central nervous system was first observed in the 4th ventricle of the brain and the central canal of the spinal cord of a rabbit by using a staining dye, chrome hematoxylin [8], and subsequently in the 3rd ventricle of a rat brain by using Trypan blue dye [9]. Fluorescent nanoparticles were injected into the lateral ventricle of a rat to detect the PV in the fourth ventricle and the spinal cord of a rat [10]. The PVS in the subarachnoid space of a rat spine [11] and in the sciatic nerve of a rat [12] has also been reported. The function of the PVS with respect to nerve regeneration and acupuncture is not yet studied [13,14].
Recently, the PVS was observed on the pia mater of a rat brain by using Alcian blue [15]. Primo vessels (PVs) and primo nodes (PNs) were found underneath the superior sagittal sinus (SSS) in the sagittal fissure of a rabbit [16]. The PV underneath the SSS was also found in a rat, and it showed a strong linear optical polarization.
Thus, an investigation as to whether the PV in the falx cerebri underneath the SSS also exists in mice and exhibits a similar polarization effect is needed. Another pending effort is to find PNs in a mouse brain. The present work reports observations of a PV underneath the SSS and its optical polarization and observations of PNs on the transverse sinuses instead of the SSS.

Animal Preparation.
Eleven adult ICR mice (female, 10 weeks old, 33 g) were purchased from Dooyeol Biotech Co., Ltd., (Seoul, Korea). Animals were housed in the laboratory animal facility at 25 ∘ C and 60% relative humidity under a 12-hour light/dark cycle. Procedures involving the animals and their care were in full compliance with current international laws and policies (Guide for the Care and Use of Laboratory Animals, National Academy Press, 1996). All surgical procedures were performed under general anesthesia (25-mg/kg Zoletil and 10-mg/kg Rompun administered by intramuscular injection).

Brain Specimen Preparation.
After the mouse had been sacrificed by overanesthetizing, the mouse was decapitated, and the head was fixed by putting it in paraformaldehyde (PFA, Sigma-Aldrich, USA) for one week in the refrigerator (2 ∘ C). The head was opened one hour before the experiment and was washed with running tap water. After the skull had been separated from the brain by using tweezers, we carefully kept the dura mater and the pia mater of the brain intact. Before peeling the dura mater off the brain, as a preparation for detection of primo nodes and primo vessels with the features of nuclei distribution, we sprayed 4 ,6-diamidino-2-phenylindole (DAPI) solution on the surface of the dura mater and the pia mater of the brain. Because the dura mater had a high density of nuclei, discerning the features of the nuclei distribution in the PVS that was covered by the dura mater was difficult. Therefore, we developed techniques to peel the dura mater off the brain meninges. In this method, we were able to discern the PNs, PVs on the pia mater of the brain.

Staining and Observations with Microscopes.
We applied DAPI and phalloidin reagents for staining of nuclei and factin molecules in the cells, respectively. After a one-hour DAPI (Invitrogen, ProLong Gold Antifade Reagent with DAPI, USA) staining, we washed the sample with phosphate based saline (PBS) three times. After that, the phalloidin (Invitrogen, Rhodamine Phalloidin, USA) staining was done for one hour, and the PBS washing was done three times.
After the samples had been washed, we dried them and poured on Neomount (M1289-10 mL, Sigma-Aldrich, USA) solution on the brain with care not to make bubbles and noise so that the Neomount would not become too thick for analysis of the primo vessels with a high-magnification microscope. We gently placed the cover glass on the sample; the cover should be maintained level for high-magnification observation.
The stained specimens were investigated with a phasecontrast microscope (BX51, Olympus, Japan) and polarization microscope (KSM-BA3, Samwon, Korea) to search for loose connective tissues, such as blood capillaries, nerve tissues, and bundles of PVs and PNs. A fluorescent microscope (MVX10, Olympus, Japan) was used to investigate the characteristic features of the nuclei and the F-actin distributions of the PVs and the PNs with DAPI and phalloidin staining, respectively. Confocal laser scanning microscopy (C1 plus, Nikon, Japan) was used to optically scan the threadlike PVs to uncover the characteristic bundle structure of the PVs.

Results
A schematic illustration and a real specimen of the mouse brain are shown in Figures 1(a) and 1(b), respectively. The falx cerebri houses the superior sagittal sinus. The PV was located in the falx cerebri and could be detected because of its strong polarization signal, as shown in Figures 1(c) and 1(d). We confirmed the representative features of a PV by staining the sample with DAPI and phalloidin to reveal the nuclei and the f-actin distributions. The results are presented in Figures 2(a), 2(b), and 2(c). The confocal laser scanning microscope image shows the presence of rod-shaped nuclei and multiple bundle of channels, as shown with the asterisk | in the lower panel of Figure 2(c).
We observed PNs at two locations in the subarachnoid space between the pia mater and the arachnoid mater of a mouse brain: the first location, named as the H-point, is under the transverse sinus at the boundary between the hemisphere and the vermis of the cerebellum. The second location, named as the P-point, is further outside along the transverse sinus near the end of the hemisphere of the cerebellum. The average distances from the H-point and the P-point to the (lambda) point were 3,763 m and 5,967 m (Figure 1(a)).
As shown in Table 1, we did experiments with eleven mice and found six PNs at the left H-point, six at the right H-point, three at the left P-point, and three at the right P-point. The average distances from the point and the average size were as follows: Remarkably PNs of fairly distinguishable shapes (Figures 3, 4, and 5) were repeatedly observed at the H-points and the P-points along the transverse sinuses.
A detailed description of a PN at the H-point is shown in Figure 3. The location in the dura mater was determined under a stereomicroscope (Figure 3(a)), and the PN specimen was observed with a phase-contrast microscope equipped for fluorescence. The observation after DAPI and phalloidin staining revealed cells and nuclei packed in the PN, as expected (Figures 3(b), 3(c), 3(d), 3(e)). A more realistic view was obtained with a confocal laser scanning microscope as presented in Figure 3(f). The PV attached to the PN showed rod-shaped nuclei aligned along the PV (Figure 3(g)).
Another case of a PN at the H-point is presented to show that its apparent shape is different from the previous shape (Figures 4(b), 4(c), and 4(d)). In this example, we exposed   the PN and its attached PV by removing the surrounding dura and pia maters (Figures 4(e) and 4(f)). The DAPI signals showed packed nuclei in the PN and rod-shaped nuclei in the PV.
A PN at the P-point was observed, as indicated with the dotted circle in Figure 5(a). Its shape was oval, and its size was 230 m × 460 m. Its phase-contrast, DAPI-stained, MVX-10, and phalloidin-stained images (Figures 5(d), 5(e), 5(f), and 5(g), resp.) were similar to those for the PNs at the H-points. A PN at the H-point is indicated with a circle in Figure 5

Discussion
The PV in the falx cerebri embedded in the dura mater under the sagittal sinus was first observed in a rabbit brain [15] and subsequently in a rat brain. The current work confirmed the presence of a similar PV in a mouse brain. The thicknesses of the PVs were 55.7 m, 56.7 m, and 31 m in the cases of rabbits, rats, and mice, respectively. The distributions of rod-shaped nuclei and the bundle structures of the multiple channels were similar.
The linear polarization of the PV that was previously observed in a rat brain was also clearly visible in the mouse  case. This optical property was useful in detecting the PNs along the transverse sinus, as shown in Figure 5.
The presence of a PN in the brain was noticed in the case of a rabbit at the confluence of the transverse sinuses, that is, near the point [15]. However, no PN was detected at a similar location in mice. In our experiment, PNs were observed at completely different positions, the left and the right H-points and the left and the right P-points along the transverse sinuses. The sizes of the PNs were 286 m × 503 m and 155 m × 248 m in rabbits and mice, respectively. The PNs of both animals were packed with many cells inside.
PNs packed with various cells were a common feature. Especially, immune cells were abundant in the PNs on the Evidence-Based Complementary and Alternative Medicine 5 The subjects were 10-week-old female ICR mice. The primo nodes had cucumber or oval shapes; short and long axes were measured.

H-point
Lambda point  Table 1  surfaces of internal organs [17] and inside lymph vessels [18]. Immunohistochemical data also suggested the presence of embryonic-like stem cells in the PNs on the surfaces of internal organs and inside the lymph vessels [18]. Furthermore, observations of cancer stem cells in the PNs of xenografted cancer mice were recently reported [19].
In the current work, we were not able to isolate and analyze the cells from the brain PNs. It remains an important question whether the PNs in brain contain abundant immune cells and/or stem cells as observed in the PNs on the surfaces of internal organs. There are also critical works against the acupuncture relevance of the PVS [20]. These medically critical features are worthy of more future work.