Figures & data
Figure 1. Generation and characterisation of the TRPM4 humanised KI rat model. (A) Schematic strategy for generating the KI rat model. (B) KI rat contains a human TRPM4 polypeptide to replace the corresponding rat sequences. The amino acid sequences used for generating M4M were circled with boxes. Filled grey box indicates the channel pore region. M4M binding epitope EPGF were highlighted in red. (C) DNA sequencing results on homozygous KI rats. (D) Genotyping of the WT (+/+), heterozygous (+/−) and homozygous (−/−) rats. (E) Western blot on brains from WT (+/+), heterozygous (+/−) and homozygous (−/−) rats using monoclonal antibody M4M that is specific for human TRPM4 [Citation10]. The Immunoblot was probed with anti-Actin antibody as the housekeeping protein.
![Figure 1. Generation and characterisation of the TRPM4 humanised KI rat model. (A) Schematic strategy for generating the KI rat model. (B) KI rat contains a human TRPM4 polypeptide to replace the corresponding rat sequences. The amino acid sequences used for generating M4M were circled with boxes. Filled grey box indicates the channel pore region. M4M binding epitope EPGF were highlighted in red. (C) DNA sequencing results on homozygous KI rats. (D) Genotyping of the WT (+/+), heterozygous (+/−) and homozygous (−/−) rats. (E) Western blot on brains from WT (+/+), heterozygous (+/−) and homozygous (−/−) rats using monoclonal antibody M4M that is specific for human TRPM4 [Citation10]. The Immunoblot was probed with anti-Actin antibody as the housekeeping protein.](/cms/asset/a33fac0c-9c7a-4e9a-8fc2-3c9d0c61397c/idrt_a_2313522_f0001_c.jpg)
Figure 2. Immunofluorescent staining of primary cultured neurons from embryonic homozygous rat brains. Neurons were treated under normoxia or hypoxia for 24 h prior to fixation. Control mouse IgG or M4M (red) were used to stain neurons that were labelled with neuronal marker MAP2 (green). Nuclei were counterstained with DAPI (blue). Scale bars: 20 µm.
Figure 3. Functional characterisation of M4M on primary cultured neurons from TRPM4 humanised rats. (A) Current voltage relationships of primary cultured neurons from wild-type (+/+) rats before (0 min) and after 7-min of hypoxic induction under the treatment of vehicle, 10 µg/mL control mouse IgG (mIgG) or 10 µg/mL M4M. Ramp protocols were applied from − 80 to + 80 mV with a holding potential at 0 mV. (B) Summary of the currents at + 80 and -80 mV. (C) current voltage relationships of primary cultured neurons from homozygous rats (−/−) expressing human TRPM4 sequences. (D) Summary of the currents at + 80 and -80 mV from C. (E) Sample images of WT (+/+) neurons before (0 min) and after 10 min of ATP depletion by 5 mM NaN3 and 10 mM 2-DG. Scale bars: 10 µm. (F) time course of membrane capacitance (Cm) during 10 min of ATP depletion in WT neurons. The Cm is normalised to baseline at 0 min. (G) Sample images of homozygous (−/−) neurons before and after 10 min of ATP depletion. Scale bars: 10 µm. (H) Time course of Cm changes during 10 min of ATP depletion in homozygous (−/−) neurons. Statistical analysis was performed by two-way ANOVA test with post hoc Bonferroni’s analysis. *p < 0.05, **p < 0.01, ***p < 0.0001, #p < 0.0001.
Figure 4. Immunofluorescent staining of TRPM4 using M4M in homozygous rats. Left MCAO was created for 3 h followed by 24-h reperfusion. (A) M4M (green) and neuronal marker, anti-NeuN (red) were used to co-stain neurons within the ipsilateral and contralateral hemispheres. (B) Co-localisation of anti-NeuN, M4M and DAPI (blue) within the ipsilateral hemisphere from A. Asterisks: M4M negative neurons. Majority of the neurons within the dotted line were positive for both NeuN and M4M. Scale bars for A–B: 100 µm. (C) Double staining of M4M (green) and endothelial marker vWF (red) on the ipsilateral hemisphere. (D) Co-localisation of vWF, M4M and DAPI (blue) within the ipsilateral hemisphere from C. Asterisks: partly colocalization of M4M and anti-vWF. (E) Double staining of M4M (green) and astrocyte marker GFAP (red) on the ipsilateral hemisphere. Asterisks: partly colocalization of M4M and GFAP. Scale bars for C–E: 50 µm.
Figure 5. Evaluating the in vivo role of M4M in a stroke reperfusion animal model. (A) Diagram showing experimental protocol for 3-h transient MCAO. M4M (100 µg) or control mouse IgG (mIgG, 100 µg) was injected intravenously (i.v. Ab) 1 h before recanalization. (B) Representative images of TTC-stained brains from homozygous (−/−), heterozygous (+/−), and wild-type (+/+) rats at 1 day after transient MCAO induction that were treated with mIgG or M4M. C, Summary of infarct volume formation of rat brains at 1 day after transient MCAO induction. n = 6 rats for M4M and mIgG treatments in wild-type and homozygous rats except for treatments in heterozygous rats (n = 7). (D) Comparison of percentage of infarct volume normalised to the whole brain region after M4M treatment among homozygous, heterozygous, and wild-type rats. (E) Assessment of motor functions by Rotarod test in homozygous rats after the treatment of M4M (n = 7), mIgG (n = 6), or vehicle (n = 6). (F) Comparison of the neurological severity scores in homozygous rats treated with vehicle (n = 6), mIgG (n = 6), and M4M (n = 7). In C, E, and F, statistical analysis was performed by two-way ANOVA with Bonferroni post hoc test, and in D by one-way ANOVA with Bonferroni post hoc test. *p < 0.05, **p < 0.01. In E and F, significance was observed in M4M versus vehicle or mIgG at day 1. After day 1, significance was found only in M4M versus mIgG.
