Extended Data Fig. 4: Essential Roles of the mLV Intraluminal Valves in Brain Fluid Drainage.
From: Piezo1 regulates meningeal lymphatic vessel drainage and alleviates excessive CSF accumulation
(a-j) Targeting Lymphatic Valves during the Developmental Phase in Neonates. Control mice (CTR) (Prox1-CreERT2; Piezo1+/+; Prox1-tdTomato) and lymphatic Piezo1 KO mice (Pz1 dLEC) (Prox1-CreERT2; Piezo1flox/flox; Prox1-tdTomato) were subcutaneously injected with Tamoxifen at a low dose (15 mg/kg) at P1 to target deletion of Piezo1 in lymphatic valves. The mice were euthanized at P21 for further analyses of lymphatic valves and vessels. (a) Microscopic images showing the presence of lymphatic valves (arrowheads) in the basal mLVs around Pterygopalatine Artery (PPA), Petrosquamous Fissure (PSF), and Sigmoid Sinus (SS). Scale bars: 500 µm. (b-d) Number of lymphatic valves in these areas was counted. n = 6 mice/group. (e) Images showing the absence of morphological changes of mLVs in mice (P21) treated with low dose Tamoxifen. Scale bars: 200 µm. The lymphatic vascular density (f) and thickness (g) in the control and Pz1 dLEC mice showed no significant differences. n = 6 mice/group (h) A brain fluid tracer (OVA-Green: Ovalbumin, Alexa Fluor™ 488 Conjugate) was i.c.m. injected at P21. The cervical and mandibular LNs were harvested and imaged 60 minutes after the tracer injection. Note dramatically diminished tracer drainages. Scale bars: 500 µm. (I, j) The relative OVA intensity in LNs was quantified. n = 10 mice/group. (k-o) Targeting Matured Lymphatic Valves in Adult Mice: Control (CTR) and Pz1 dLEC mice (6 months old) were i.p. injected with Tamoxifen at a low dose (15 mg/kg, twice, three days apart). After 6 days from the first tamoxifen administration, a brain fluid tracer (OVA-Green: Ovalbumin, Alexa Fluor™ 488 Conjugate) was i.c.m. injected, and the mice were euthanized to analyze lymphatic valves and brain fluid drainage. Lymphatic valves (arrowheads) around Pterygopalatine Artery (PPA) and Petrosquamous Fissure (PSF) were imaged (k) and their numbers were counted (I, m). n = 6 mice/group. (n) After 60 minutes from the tracer injection, the cervical and mandibular LNs were harvested and imaged. Scale bars: 500 µm. (o) The relative intensity of OVA-Green in LNs was quantified. n = 6 mice/group. Each data point represents the sum of the fluorescence intensity of LNs on the right and left sides of a single mouse (h, n). Statistics: (b, f) two-tailed Mann-Whitney U test. (c, d, g, i, j, l, m, o) two-tailed t-test. Statistics: two-tailed t-test. Data are presented as mean values +/- SEM.
