Figures and data in Loss of aquaporin-4 results in glymphatic system dysfunction via brain-wide interstitial fluid stagnation

Version of Record: March 8, 2023
Accepted Manuscript: February 9, 2023
Accepted Manuscript: February 9, 2023

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Ryszard Stefan Gomolka

Lauren M Hablitz

Humberto Mestre

Michael Giannetto

Ting Du

Natalie Linea Hauglund

Lulu Xie

Weiguo Peng

Paula Melero Martinez

Maiken Nedergaard

Yuki Mori

(2023)
Loss of aquaporin-4 results in glymphatic system dysfunction via brain-wide interstitial fluid stagnation

eLife 12:e82232.

https://doi.org/10.7554/eLife.82232
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8 figures, 4 tables and 2 additional files

Figures

Figure 1

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3D-CISS MRI, CSF and interstitial space volumetry *in vivo*.

Overlaid 3D surface images of the co-registered and averaged 3D-CISS brain volumes, and whiskers-box plots comparing (A) the brain volumes and (B) segmented CSF space volumes from 6 WT and 5 AQP4 KO …

Figure 1—source data 1 3D-CISS CSF space volumetry in vivo - source data.: https://cdn.elifesciences.org/articles/82232/elife-82232-fig1-data1-v3.xlsx
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Figure 1—source data 2 Brain water content ex vivo - source data.: https://cdn.elifesciences.org/articles/82232/elife-82232-fig1-data2-v3.xlsx
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Figure 1—source data 3 CSF production in vivo - source data.: https://cdn.elifesciences.org/articles/82232/elife-82232-fig1-data3-v3.xlsx
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Figure 1—source data 4 ISF space volume estimation with TMA - source data.: https://cdn.elifesciences.org/articles/82232/elife-82232-fig1-data4-v3.xlsx
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Figure 2

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Diffusion-weighted MRI *in vivo* and *ex vivo*, vascularity, and AQP4 cellular surface expression *ex vivo*.

Radar plots showing statistical significances for the differences between the slow diffusion measures among 15 parenchymal ROI assessed, for average ADC (A) and D, and in (B) 3 diffusion-encoding …

Figure 2—source data 1 DWI and IVIM-DWI estimates in vivo - source data.: https://cdn.elifesciences.org/articles/82232/elife-82232-fig2-data1-v3.xlsx
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Figure 2—source data 2 DWI, IVIM-DWI, and T1 estimates in the phantom - source data.: https://cdn.elifesciences.org/articles/82232/elife-82232-fig2-data2-v3.xlsx
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Figure 2—source data 3 Immunohistochemistry: vascular labeling - source data.: https://cdn.elifesciences.org/articles/82232/elife-82232-fig2-data3-v3.xlsx
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Figure 2—source data 4 Immunohistochemistry: AQP4 channel labeling - source data.: https://cdn.elifesciences.org/articles/82232/elife-82232-fig2-data4-v3.xlsx
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Figure 3

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Figure 3—source data 1 DCE-MRI in vivo - source data.: https://cdn.elifesciences.org/articles/82232/elife-82232-fig3-data1-v3.xlsx
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Appendix 1—figure 1

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Supplementary results for MR CSF space volumetry and MR diffusion evaluation.

(A) Whiskers-box plot comparison of segmented CSF regions, where no statistical difference between KO and WT was found, along with overlaid 3D surface images of separate compartments (colored) from …

Appendix 1—figure 2

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Supplementary results for dynamic contrast-enhanced MRI *in vivo* and correlational analysis.

(A) 3D multiplanar reconstructions of dynamic-contract enhanced (DCE) MRI – midsagittal, parasagittal, and lateral along with orthogonal axial and coronal slices from mean 3D FISP images from 5 AQP4 …

Appendix 1—figure 3

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Schematic flow chart of the algorithm for the 3D-CISS-based CSF space volumetry and cisternography in *Aqp4(-/-) (KO)* and *Aqp4(+/+) (WT)* mice, including initial preprocessing (bias field correction and brain extraction) of the computed 3D-CISS volume (A) and subsequent automatic CSF space segmentation (B), based on representative 3D-CISS volume from a single animal.

Author response image 1

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Inverse Laplace transform (ILT) performed using INSPECT algorithm assuming presence of 2 spectral components.

Presences of fast diffusivities (>10 um²/ms, perfusion-like) components was confirmed in aggregated ADC spectra from 3 regions analyzed (A-C, see ‘Distribution diffusion-encoding direction-wise’). …

Author response image 2

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3D-CISS-based regional brain morphometry in *Aqp4* (+/+) and *Aqp4* (-/-) mice brains.

Legend: OLF-olfactory, CA-cingulate, VIS (V1)-visual, SS (S1)-somatosensory, M-motor; AUD-auditory, HIP-hippocampus, PERI-perirhinal, TH-thalamus, HAB-habenula, HY-hypothalamus, MB-midbrain, …

Tables

Table 1

Summary of demographic characteristics of the animals used in 8 experiments performed, and (B) details of MRI protocols and acquisition parameters for evaluation of the brain CSF and ISF spaces, employed in the current study.

Legend: CryoProbe – cryogenically-cooled MR coil; TA - time of acquisition; Tx/Rx - transmit/receive; TR - time to repetition; TE - time to echo; FA - flip angle; FOV - field of view; Av. / Rep. – …

A	No.of animals			Age [weeks]		Body weight [g]		Respiration [bpm]
	KO	WT	Male [%]	Overall (mean ±SD)	p	Overall (mean ±SD)	p	Overall (mean ±SD)	p
3D-CISS volumetry	5	6	72.7	13.8±1.8	NS	27.3±2.1	NS	176±30	NS
MR-DWI	6	6	41.7	10.4±0.7	NS	22.4±3.0	NS	175±11	NS
DCE-MRI	5	6	45.5	13.4±1.6	NS	27.3±2.2	NS	182±14	NS
AQP4 expression	-	4	50	10.0±0.0	NS	25–30	N/A	-	-
Vascular density	6	6	0	14.6±1.0	NS	24.3±4.6	NS	-	-
CSF production	5	6	45.5	15.4±0.5	NS	28.7±4.2	NS	-	-
ISF space volume estimation using TMA	8	20	55	10.0±0.0	NS	25–30	N/A	-	-
Brain water content	3	5	62.5	12.0±1.0	NS	25–30	N/A	-	-

B	MR sequences and acquisition parameters
Sequence (Tx/Rx coil; slice orientation)	TR [ms]	TE [ms]	FA [deg]	Av./Rep.	Voxel size [mm³](interpolation)	Bandwidth [Hz/pix]	Matrix size	TA
MR CSF space volumetry
3D-TrueFISP (CryoProbe; sagittal)	5.2	2.6	50	2	0.033×0.033×0.033 (2.0×1.6×1.0)	260	19.2×12.8×12.8	27 min
MR-DWI (in vivo and ex vivo) (δ=3 and Δ=10ms for gradient duration and separation times) *(b-values (Av.>1)): 40, 50, 59, 70, 92, 113, 165, 197, 238, 342, 445, 649, 854, 1057 (2), 1564 (2), 2071 (2), 3081 (2) s²/mm
2D-EPI (volumetric; axial)	3600	30	90	3	0.15×0.15×0.5 (0.2 mm gap, 16 slices)	3307	16.2×14.4×11.2	20–30 min (respiratory-gated)
DCE-MRI via CM-injection
3D-FISP (CryoProbe; sagittal)	3.26	1.63	15	1	0.1×0.1×0.1	781	19.2×12.8×12.8	90 min
Microbeads phantom ex vivo - T1 mapping (VTR: 12000, 9000, 6500, 4000, 2000, 1000, 800, 500, 300, 100, 80, 50, 15ms) (VFA: 45° and 90°)
2D-RARE (volumetric; axial)	VTR	3.1	90	2	0.1×0.1×3.0	671	16.2×16.2×1	3 h 20 min
2D-RARE (volumetric; axial)	12000	3.1	VFA	3	0.1×0.1×3.0	671	16.2×16.2×1	1 hr 40 min

*

Presented averages of measured values from 3 diffusion encoding directions are slightly higher than the set-up, due to gradient preparation time.

Table 2

Summary of findings for (A) average ADC and D-IVIM, (B) direction-wise MR diffusion and pseudodiffusion among 21 ROI assessed, along with statistical scoring.

Asterisks reflect ‘p’ significance values from the nonparametric Mann-Whitney test comparing diffusion measures between KO vs. WT animals ROI-wise (total n=21, balanced groups), along with the …

A	ROI	Average ADC		Average D-IVIM		IVIM
	ROI	Finding	Significance	Finding	Significance	D, Fp,Fp x D
Cerebral cortex	*OLF*	KO>WT	*	-	NS	-
	*CA / RSP*	-	NS	-	NS	-
	*VIS*	-	NS	-	NS	-
	SS	KO>WT	*	KO>WT	*	-
	*AUD*	KO>WT	*	KO>WT	*	-
	*HIP*	KO>WT	**	KO>WT	*	-
	*PERI*	-	NS	-	NS	-
Brain stem	TH	KO>WT	**	KO>WT	**	-
	*HAB*	-	NS	-	NS	-
	HY	-	NS	-	NS	-
	MB	KO>WT	*	KO>WT	*	-
	*PAG*	KO>WT	*	-	NS	-
	HB	KO>WT	**	KO>WT	**	-
Cerebral nuclei and tracts	CP	KO>WT	**	KO>WT	*	-
Cerebral nuclei and tracts	WM	KO>WT	*	KO>WT	*	-
CSF space	3V	-	NS	-	NS	-
	LV	-	NS	-	NS	-
	4V	-	NS	-	NS	-
	*PCS*	-	NS	-	NS	-
	*CoW*	-	NS	-	NS	-
Cerebellum	CB	-	NS	-	NS	KO>WT Fp = 0.0649
B	ROI	IVIM						ADC / D
		Direction Z(cranio-caudal)		Direction X(bilateral)		Direction Y(ventral-dorsal)		Direction Z (cranio‐caudal)		Direction X (bilateral)		Direction Y (ventral‐dorsal)
		Finding	Signif.	Finding	Signif.	Finding	Signif.	Finding	Signif.	Finding	Signif.	Finding	Signif.
Cerebral cortex	*OLF*	-	NS	Fp x D* KO>WT	*	-	NS	KO>WT	*/	-	NS	-	NS
	*CA / RSP*	D* KO>WT	*	-	NS	-	NS	-	NS	-	NS	-	NS
	*VIS*	-	NS	-	NS	-	NS	-	NS	KO>WT	*/-	KO>WT	*/-
	SS	-	NS	-	NS	-	NS	KO>WT	-/*	KO>WT	*/ P=0.056*	KO>WT	*/-
	*AUD*	-	NS	-	NS	-	NS	KO>WT	/	KO>WT	*/	KO>WT	/
	*HIP*	-	NS	-	NS	-	NS	KO>WT	/	KO>WT	*/-	KO>WT	*/
	*PERI*	D* KO>WT	**	-	NS	-	NS	-	NS	KO>WT	*/-	-	NS
Brain stem	TH	-	NS	-	NS	-	NS	KO>WT	/	KO>WT	*/-	KO>WT	*/-
	*HAB*	-	NS	-	NS	-	NS	-	NS	-	NS	-	NS
	HY	-	NS	-	NS	-	NS	-	NS	-	NS	-	NS
	MB	-	NS	-	NS	-	NS	KO>WT	-/*	KO>WT	*/-	KO>WT	**/-
	*PAG*	-	NS	-	NS	-	NS	KO>WT	/*	KO>WT	**/-	KO>WT	*/-
	HB	-	NS	-	NS	-	NS	KO>WT	/	KO>WT	**/-	KO >WT	/
Cerebral nuclei and tracts	CP	-	NS	-	NS	-	NS	KO>WT	/	KO>WT	*/-	-	NS
Cerebral nuclei and tracts	WM	D* KO>WT	**	-	NS	-	NS	-	NS	-	NS	KO>WT	*/-
CSF space	3V	-	NS	Fp / Fp x D* KO>WT	** / *	-	NS	-	NS	-	NS	-	NS
	LV	-	NS	-	NS	-	NS	-	NS	-	NS	-	NS
	4V	-	NS	-	NS	-	NS	-	NS	-	NS	-	NS
	*PCS*	-	NS	-	NS	-	NS	-	NS	-	NS	-	NS
	*CoW*	-	NS	-	NS	-	NS	-	NS	-	NS	-	NS
Cerebellum	CB	-	NS	-	NS	-	NS	-	NS	-	NS	-	NS

Table 3

DCE-derived parameters from 21 ROI (matching those analyzed by means of MR-DWI) in 5 KO and 6 WT littermate mice, along with associated nonparametric pair-wise statistics using a two-tailed Wilcoxon signed-rank test and median ± standard deviation (SD) values strain-wise.

Legend: Mean AUC – mean from areas under the DCE curves along with associated p-statistical values (p-stat) from nonparametric Mann–Whitney U-test ROI-wise; Duration – duration of significantly …

		^#WT vs. KO time-series different?	Mean AUC [a.u.]			Arrival time [min]			Time- to-peak [min]			Peak intensity [a.u.]			Interstitial tracer accumulation time [min]				Duration [min]
ROI			Aqp4(+/+)	Aqp4(-/-)	p-stat	Aqp4(+/+)	Aqp4(-/-)	Δ	Aqp4(+/+)	Aqp4(-/-)	Δ	Aqp4(+/+)	Aqp4(-/-)	rel. Δ [%]	Aqp4(+/+)		Aqp4(-/-)		Aqp4(+/+)	Aqp4(-/-)	rel. Δ [%]
ROI			Aqp4(+/+)	Aqp4(-/-)	p-stat	Aqp4(+/+)	Aqp4(-/-)	Δ	Aqp4(+/+)	Aqp4(-/-)	Δ	Aqp4(+/+)	Aqp4(-/-)	rel. Δ [%]	Start	End	Start	End	Aqp4(+/+)	Aqp4(-/-)	rel. Δ [%]
*Cortical*	*OLF*	NS	7207	5619	NS	6	6	0	61	52	9	106	79	*25.5*	43	80	39	70	38	32	–15.8
	*CA/RSP*	**	1662	616	**	7	7	0	89	90	-1	27	12	*55.6*	59	90	66	90	32	25	–21.9
	*VIS*	**	1699	529	**	7	8	-1	89	90	-1	33	9	*72.7*	54	90	73	90	37	18	–51.4
	SS	NS	537	438	NS	6	7	-1	20	16	4	8	7	*12.5*	12	32	11	25	21	15	–28.6
	*AUD*	NS	454	340	NS	7	7	0	89	89	0	10	5	*50.0*	67	90	N/A	N/A	24	*N/A*	<-100
	*HIP*	**	3258	1622	*	5.5	7.5	-2	63	81	–18	46	22	*52.2*	45	90	74	90	46	17	–63.0
	*PERI*	P=0.052	3513	2659	NS	4	5	-1	83	90	-7	59	44	*25.4*	53	90	57	90	38	34	–10.5
*Brain stem*	TH	*	3471	2557	0.052	2–3	2–3	0	82	90	-8	61	45	*26.2*	53	90	58	90	38	33	–13.2
	*HAB*	NS	2379	1992	NS	9	11	-2	89	90	-1	45	36	*20.0*	54	90	58	90	37	33	–10.8
	HY	NS	7908	6766	NS	4	4	0	39	34	5	123	105	*14.6*	23	59	21	54	37	34	–8.1
	MB	*	6125	4013	*	3	4	-1	66	83	–17	87	56	*35.6*	42	84	53	90	43	38	–11.6
	*PAG*	NS	25740	22474	NS	8	10	-2	43	38	5	350	311	*11.1*	35	75	31	61	41	31	–24.4
	HB	NS	9291	8124	NS	2–3	2–3	0	42	38	4	133	114	*14.2*	28	66	27	61	39	35	–10.3
*Nuclei / tracts*	CP	NS	1736	1167	NS	4	8	-4	89	90	-1	39	25	*35.9*	54	90	58	90	37	33	–10.8
*Nuclei / tracts*	WM	**	6953	5445	**	4.5	6.5	-2	51	53	-2	105	80	*23.8*	37	73	38	69	37	32	–13.5
*CSF space*	CM	NS	23276	19903	NS	1	1	0	24	24	0	397	339	*14.6*	15	50	16	47	36	32	–11.1
	PCS	NS	15163	12498	NS	2–3	2–3	0	41	31	10	224	187	*16.5*	23	59	20	50	37	31	–16.2
	3V	NS	12228	10441	NS	5	6	-1	45	46	-1	180	151	*16.1*	34	76	33	61	43	29	–32.6
	LV	NS	8288	6555	NS	6	11	-5	90	90	0	157	123	*21.7*	54	90	58	90	37	33	–10.8
*Caudal*	SSS	*	5832	1430	**	1	3	-2	64	88	–24	77	22	*71.4*	38	90	75	90	53	16	–69.8
*Caudal*	CB	NS	7369	6151	NS	6	7	-1	47	59	–12	97	80	*17.5*	29	75	31	67	47	37	–21.3
MEDIAN ±SD			6125±1653^¶	4013±1306^¶		5.0±2.2	6.5±2.8		63±23	81±27		56	87	24±19					37±7	32±7
*KO vs. WT difference* *(Wilcoxon signed-rank test)*			WT >KO, p<0.0001			WT <KO, p<0.01			WT ~KO, p=0.2971			WT >KO, p<0.0001							WT >KO, P<0.0001

Table 4

Descriptive summary of findings presented in the current study.

Bold italic font highlights the regions of the largest differences found between KO and WT animals, by means of 3 MRI and 5 physiological and histological assessment methods applied.

Magnetic resonance imaging in vivo
Measurement		General findings in KO compared to WT	Region of largest difference
non-invasive	3D cisternography	- 5–10% larger brain volumes - 22–29% smaller CSF space / brain volume ratio	ventricular space, 3^rd ventricle
	2D diffusion-weighted imaging	5–6% higher ADC and D	(difference present for average and in all diffusion directions) - thalamus, hindbrain, periaqueductal gray regions, auditory cortex and hippocampus
	2D diffusion-weighted imaging	Higher Fp and Fp × D* only in the 3^rd ventricle
invasive	Dynamic CSF tracer imaging via cisterna magna`	reduced parenchymal tracer influx and evacuation	- influx: cortical ROI, hippocampus - efflux: superior sagittal sinus

Physiological and histological measurements
Measurement		General findings in KO compared to WT	Region of largest difference
ex vivo and histology	Brain water content	~6% larger brain water content
	AQP4 expression	(only WT) heterogenous AQP4 expression in the brain	largest expression in the thalamus, hippocampus, habenula
	Vascular density	similar vascular density to WT	trend for larger vascular density in the thalamus and olfactory area
*in vivo*	CSF production	similar CSF production to WT
*in vivo*	Real-time ionophoresis TMA	ISF space volume larger

Additional files

MDAR checklist: https://cdn.elifesciences.org/articles/82232/elife-82232-mdarchecklist1-v3.docx
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Source data 1 Aggregated data set, including all source data.: https://cdn.elifesciences.org/articles/82232/elife-82232-data1-v3.zip
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Figures

3D-CISS MRI, CSF and interstitial space volumetry in vivo.

Figure 1—source data 1

Figure 1—source data 2

Figure 1—source data 3

Figure 1—source data 4

Diffusion-weighted MRI in vivo and ex vivo, vascularity, and AQP4 cellular surface expression ex vivo.

Figure 2—source data 1

Figure 2—source data 2

Figure 2—source data 3

Figure 2—source data 4

Dynamic contrast-enhanced MRI in vivo (Figure 3—source data 1).

Figure 3—source data 1

Supplementary results for MR CSF space volumetry and MR diffusion evaluation.

Supplementary results for dynamic contrast-enhanced MRI in vivo and correlational analysis.

Inverse Laplace transform (ILT) performed using INSPECT algorithm assuming presence of 2 spectral components.

3D-CISS-based regional brain morphometry in Aqp4 (+/+) and Aqp4 (-/-) mice brains.

Tables

Summary of demographic characteristics of the animals used in 8 experiments performed, and (B) details of MRI protocols and acquisition parameters for evaluation of the brain CSF and ISF spaces, employed in the current study.

Summary of findings for (A) average ADC and D-IVIM, (B) direction-wise MR diffusion and pseudodiffusion among 21 ROI assessed, along with statistical scoring.

DCE-derived parameters from 21 ROI (matching those analyzed by means of MR-DWI) in 5 KO and 6 WT littermate mice, along with associated nonparametric pair-wise statistics using a two-tailed Wilcoxon signed-rank test and median ± standard deviation (SD) values strain-wise.

Descriptive summary of findings presented in the current study.

Additional files

MDAR checklist

Source data 1

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3D-CISS MRI, CSF and interstitial space volumetry in vivo.

Figure 1—source data 1

Figure 1—source data 2

Figure 1—source data 3

Figure 1—source data 4

Diffusion-weighted MRI in vivo and ex vivo, vascularity, and AQP4 cellular surface expression ex vivo.

Figure 2—source data 1

Figure 2—source data 2

Figure 2—source data 3

Figure 2—source data 4

Dynamic contrast-enhanced MRI in vivo (Figure 3—source data 1).

Figure 3—source data 1

Supplementary results for MR CSF space volumetry and MR diffusion evaluation.

Supplementary results for dynamic contrast-enhanced MRI in vivo and correlational analysis.

Inverse Laplace transform (ILT) performed using INSPECT algorithm assuming presence of 2 spectral components.

3D-CISS-based regional brain morphometry in Aqp4 (+/+) and Aqp4 (-/-) mice brains.

Summary of demographic characteristics of the animals used in 8 experiments performed, and (B) details of MRI protocols and acquisition parameters for evaluation of the brain CSF and ISF spaces, employed in the current study.

Summary of findings for (A) average ADC and D-IVIM, (B) direction-wise MR diffusion and pseudodiffusion among 21 ROI assessed, along with statistical scoring.

DCE-derived parameters from 21 ROI (matching those analyzed by means of MR-DWI) in 5 KO and 6 WT littermate mice, along with associated nonparametric pair-wise statistics using a two-tailed Wilcoxon signed-rank test and median ± standard deviation (SD) values strain-wise.

Descriptive summary of findings presented in the current study.

MDAR checklist

Source data 1