Characterising spinal cerebrospinal fluid flow in the pig with phase-contrast magnetic resonance imaging

doi:10.21203/rs.3.rs-1966542/v1

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Research Article

Characterising spinal cerebrospinal fluid flow in the pig with phase-contrast magnetic resonance imaging

https://doi.org/10.21203/rs.3.rs-1966542/v1

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Journal Publication

published 18 Jan, 2023

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Background

Detecting changes in pulsatile cerebrospinal fluid (CSF) flow may assist clinical management decisions, but spinal CSF flow is relatively understudied. Traumatic spinal cord injuries (SCI) often cause spinal cord swelling and subarachnoid space (SAS) obstruction, potentially causing pulsatile CSF flow changes. Pigs are emerging as a favoured large animal SCI model; therefore, the aim of this study was to characterise CSF flow along the healthy pig spine.

Methods

Phase-contrast magnetic resonance images (PC-MRI), retrospectively cardiac gated, were acquired for fourteen laterally recumbent, anaesthetised and ventilated, female domestic pigs (22–29 kg). Axial images were obtained at C2/C3, T8/T9, T11/T12 and L1/L2. Dorsal and ventral SAS regions of interest (ROI) were manually segmented. CSF flow and velocity were determined throughout a cardiac cycle. Linear mixed-effects models, with post hoc comparisons, were used to identify differences in peak systolic/diastolic flow, and maximum velocity (cranial/caudal), across spinal levels and dorsal/ventral SAS. Velocity wave speed from C2/L3 to L1/L2 was calculated.

Results

PC-MRI data were obtained for 11/14 animals. Pulsatile CSF flow was observed at all spinal levels. Peak systolic flow was greater at C2/C3 (dorsal: -0.37 ± 0.17 mL/s, ventral: -0.19 ± 0.17 mL/s) than T8/T9 (dorsal: -0.04 ± 0.03 mL/s, ventral: -0.09 ± 0.09 mL/s) in both SAS regions (p < 0.001), and not different between thoracolumbar levels (p > 0.05). Peak diastolic flow was greater at C2/C3 (0.179 ± 0.06 mL/s) compared to T8/T9 (0.04 ± 0.02 mL/s) dorsally (p < 0.001), but not different ventrally (p = 1.000). Caudal maximum velocity at C2/C3 was greater than at thoracolumbar levels dorsally and ventrally (p < 0.001), but not different ventrally for cranial maximum velocity (p > 0.05). Diastolic velocity wave speed was 1.41 ± 0.39 m/s dorsally and 1.22 ± 0.21 m/s ventrally, and systolic velocity wave speed was 1.02 ± 0.25 m/s dorsally and 0.91 ± 0.22 m/s ventrally.

Conclusions

In anaesthetised and ventilated domestic pigs, spinal CSF has lower pulsatile flow and slower velocity wave propagation, compared to humans. This study provides baseline CSF flow at spinal levels relevant for future SCI research in this animal model.

CEREBROSPINAL FLUID

PULSATILE

SPINE

FLOW

VELOCITY

PHASE-CONTRAST MAGNETIC RESONANCE IMAGING

PIG

Cerebrospinal fluid (CSF) cushions the brain and spinal cord from mechanical injury, acts as a critical transport medium for nutrients and metabolites to maintain a homeostatic environment for neuronal cells, and provides a drainage system for interstitial fluid from the central nervous system (CNS) (1). Although the CSF system remains relatively poorly understood, the diagnostic (2–6) and therapeutic (7, 8) potential of CSF dynamics have become increasingly apparent. Free-flowing CSF is vital to brain and spinal cord health, and changes to CSF flow may adversely affect fluid balance and metabolism (9–11).

CSF flow in humans is predominantly oscillatory, with cranial and caudal pulsations along the spinal cord axis synchronous with the cardiac and respiratory cycles (12). CSF pulsations can be measured with phase-contrast magnetic resonance imaging (PC-MRI). A bipolar gradient is applied in the pulse sequence to produce a phase shift, which is directly proportional to proton velocity in the direction of the axis (13). PC-MRI of CSF flow in the cerebral aqueduct and at the cranio-cervical junction are used to assist in the diagnosis of hydrocephalus (2–4) and Chiari malformation (5, 6, 14). Although CSF dynamics in the spinal column may provide similar diagnostic capabilities, the spinal region is relatively understudied. Few studies have characterised normal CSF dynamics along the spine in healthy humans (15–18) and non-human primates (NHP) (19). Other studies have found that obstructions to the intrathecal space may cause changes to CSF dynamics in the context of cervical myelopathy (20–22) and in neurogenic claudication in lumbar spinal stenosis (23).

Traumatic spinal cord injury (SCI) may alter CSF flow due to the swollen spinal cord obstructing the spinal subarachnoid space (SAS) (24, 25), and one human study has demonstrated CSF velocity changes at the level of injury in cervical SCI patients up to two years after injury (26). Pigs are emerging as a favoured large animal model for SCI research (27). The more human-like size of the porcine spinal column (28), spinal cord, and intrathecal space (29, 30) compared to rodents, provides the opportunity to study CSF dynamics using clinical imaging modalities and in-dwelling pressure sensors. It is important to examine normal spinal CSF flow in the pig to provide a benchmark for future investigations of SCI. The aim of this study was to characterise normal CSF flow along the pig spine, examining two locations proximal, and two locations distal, to thoracic level T10, which is the standard spinal level for pre-clinical contusion models of SCI (31).

Animal Ethics:

This project was approved by the South Australian Health and Medical Research Institute Animal Ethics Committee (SAM 243 and SAM-22-031) and conducted in accordance with the Australian National Health and Medical Research Council Code of Care and Use of Animals for Scientific Purposes (32).

Animals:

Magnetic resonance images (MRI) of fourteen healthy, female, Large White Landrace cross pigs (22–29 kg at imaging) were obtained. The animals were acclimatised in a purpose-built facility for 7 − 10 days prior to imaging. During this time they were provided enrichment toys, access to water, twice daily food rations, and administered once daily prophylactic antibiotics (Trimidine™ 0.15gm/10kg; Sulfadimidine 430 mg, Trimethoprim 86 mg). After imaging was completed, animals were recovered from anaesthetic, and maintained for further approved procedures on the same ethics protocol that are not reported herein, after which they were humanely killed.

Anaesthetic Protocol:

The animals were fasted overnight prior to anaesthesia and pre-medicated with medetomidine (0.02 mg/kg) and ketamine (7.5 mg/kg). Anaesthesia was induced using ketamine (11 mg/kg) and propofol (2 mg/kg), maintained with an intravenous protocol of ketamine (5.0–8.0 mg/kg/hr), propofol (2.0–6.0 mg/kg/hr), and fentanyl (8.0–15.0 ug/kg/hr) and titrated as necessary towards the end of the imaging session. A propofol bolus (2.0 mg/kg) was administered when vital signs and/or physical indications suggested lightening of anaesthesia. Anaesthetics were administered via a venous catheter that was placed in the external jugular vein via surgical cut-down immediately prior to the MRI. The animals were intubated, ventilated at 17–21 breaths/min with 270–400 mL/min of oxygen, and heart rate, oxygen saturation and end-tidal carbon dioxide were monitored.

Magnetic Resonance Imaging:

MRIs were performed on a 3T scanner (Siemens, Magnetom Skyra, Germany). Animals were placed in left lateral recumbency with two 18-channel body coils (Siemens, Germany) wrapped over their torso and neck. T2-weighted TSE (turbo spin echo) images of the cervical/thoracic and thoracic/lumbar spinal regions were acquired with the following parameters: sagittal slice thickness 4 mm, in-plane resolution 1.302 mm/pixel, repetition time (TR) 3500 ms, echo time (TE) 98 ms, 2 averages, 146° flip angle and 384 x 384 acquisition matrix. These images were used during scanning to prescribe the PC-MRI axial locations and orientations, and post-processed to measure spinal length (see below).

Phase Contrast Magnetic Resonance Imaging Acquisition:

PC-MRI acquisition was performed with retrospective cardiac gating using a pulse oximeter attached to the tail. At spinal levels C2/C3, T8/T9, T11/T12 and L1/L2, a single, axial slice orthogonal to the spinal cord in the sagittal and coronal planes, aligned with the centre of the adjacent intervertebral disc, was acquired (Fig. 1A-F). Cranial flow was positively encoded (white pixels) and the number of acquired cardiac phases ranged from 14 to 30, selected according to the target R-R interval. Acquisition duration per spinal level was between 3 and 9 minutes. PC-MRI sequence parameters included: field of view, 140 x 140 mm; TR, 46.43–59.84 ms; TE, 8.01 − 9.41ms; in-plane resolution, 0.5469 x 0.5469 mm; slice thickness 5 mm; anterior-posterior phase-encoding direction; 15° flip angle. Encoding velocities (V_ENC) of 4 cm/s and 6 cm/s were selected during a pilot study and run consecutively at each spinal level; however, one animal was also scanned with a V_ENC of 2 cm/s at each spinal level. Following completion of the first ten animals, aliasing was detected in some animals at C2/C3 in the V_ENC = 6 cm/s scans; therefore, for the last four animals (P011-P014), V_ENC of 6 cm/s, 8 cm/s and 10 cm/s were used at that level.

Phase Contrast Magnetic Resonance Imaging Post-Processing:

Post-processing of CSF flow images was undertaken by a single investigator (MB) using Segment software (Version 3.2, Medviso, Lund, Sweden) (33). At each spinal level, regions of interest (ROI) were manually defined at the border of the CSF flow signal in the SAS, on the image that displayed the greatest contrast between CSF flow signal and the surrounding tissues. ROIs were defined separately in the ventral and dorsal SAS (referred to as the SAS region). The dorsal region was split into two ROIs when distinct compartments were observed (Additional file 1: Figure S1). In the Segment software, pixel selection for flow estimation included only those pixels with their centroid inside the contour of the ROI (Fig. 1G). The ROIs were propagated to each image in the stack. To assess intra-rater reliability, repeat ROIs were completed two months later by the same investigator, on ten randomly selected PC-MRIs, across all spinal levels and animals.

Eddy current offset correction with time-dependent linear compensation was applied. Each ROI was inspected for aliasing artefact (i.e., maximum velocity exceeding the acquisition V_ENC), and aliasing was corrected with phase-unwrapping which was confirmed visually by examining the pixel-by-pixel velocity in each image. The following parameters were derived: CSF velocity (cm/s) in each ROI for each cardiac phase acquired; net CSF flow (mL/s) in each ROI for each cardiac phase acquired; maximum cranial and caudal velocity (single pixel; cm/s) in each SAS region, over the cardiac cycle.

These data were further processed using a custom MATLAB program (Version R2020a, Mathworks Inc, Natick, MA). For dorsal SAS with two ROIs, net flow data were summed, and mean velocity were averaged, to provide a single dorsal value for each outcome. Net flow and mean velocity versus time data for each cycle were interpolated to 100 points using a cubic spline, and expressed as a percentage of the cardiac cycle to normalise these data by heart rate. The following parameters were derived: peak systolic (caudal direction) and peak diastolic (cranial direction) net flow; and, mean velocity, defined as the mean velocity of all pixels within the ROI, which was used to calculate the time to peak mean velocity (systolic and diastolic; from t = 0 in the cardiac cycle).

Velocity Wave Propagation Speed

CSF velocity wave speed (VWS) (34) was estimated for each animal as the distance between the C2/C3 and L1/L2 spinal levels divided by the time taken for the velocity wave (peak systolic, peak diastolic) to travel that distance. The distance from C2/C3 to the L1/L2 intervertebral disc was measured across both T2 FSE scans (summed distance from C2/C3 to T11/T12, and T11/T12 to L1/L2) with a spline placed along the centre of the spinal cord on a sagittal view, using Materialise Mimics software (Version 22.0, Materialise, NV). For the diastolic velocity wave, the difference between time to peak mean diastolic velocity at C2/C3 and L1/L2 was calculated. For the systolic velocity wave, the difference between cardiac cycle duration and the time to peak mean systolic velocity at C2/C3 was added to the time to peak systolic velocity at L1/L2, then the difference between time to peak mean systolic velocity between the two levels was calculated.

Statistical Analyses:

All statistical analyses were performed with SPSS (version 26, IBM Corporation, Armonk, NY). Intra-rater reliability of the ROI area, and the corresponding peak diastolic and systolic flow, was determined using intraclass correlation coefficients (ICC) of the absolute agreement between the repeated ROI segmentations. ICC values > 0.8 were defined as almost perfect agreement (35). Normality and homogeneity of variance were assessed for peak systolic flow, peak diastolic flow, and maximum cranial and caudal CSF velocity data using Shapiro-Wilk and Levene’s tests. Linear mixed-effects models (LMMs) were developed to identify if there was an effect of the spinal level, SAS region (ventral and dorsal ROI) and the spinal level*SAS region interaction, on peak systolic and diastolic flow, and maximum cranial and caudal CSF velocity. Post-hoc pairwise comparisons, with Bonferroni correction, were performed for LMMs with significant interactions (significance level of α = 0.05). Experimental data is presented as mean ± standard deviation (SD), and LMM outcomes are presented as estimated marginal mean differences with the corresponding 95% confidence interval (CI).

Complete CSF flow data were obtained for 11 of 14 animals. CSF flow was not detected at any spinal level in 3 animals (P005, P011, and P013). In one animal (P003), CSF flow was not detected at T8/T9 and T11/T12. CSF flow was not detected in the dorsal SAS at T8/T9 in 4 of 11 animals (Additional file 2: Table S1). When CSF flow was not detected, the spinal level and/or SAS region was excluded from all analyses and plots. Two animals (P004 and P012) had abnormal cardiac gating; data for these animals were excluded from temporal analyses and plots (Additional file 3: Figure S2), but included in peak net flow values since the diastolic and systolic extremes were maintained. The mean heart rate for the duration of the scans was 101 ± 27 bpm (Additional file 4: Table S2).

For all levels except C2/C3, the dorsal ROIs were only slightly smaller in area than the ventral ROIs (Table 1). ROI area and associated peak diastolic and systolic flow, in the dorsal SAS and ventral SAS, demonstrated excellent intra-rater reliability and almost perfect agreement between the two measurements (Table 1).

Table 1

ROI area (mean ± SD) for all animals with detected flow signal (n = 11), and intraclass correlation coefficients (ICC) for ROIs manually defined two months apart by the same investigator using a sample of ten PC-MRIs. ROI = region of interest, SAS = subarachnoid space, PC-MRI = phase contrast magnetic resonance imaging.
	Dorsal SAS	Ventral SAS
ROI area for C2C3 (mm²)	14.9 ± 4.9	10.1 ± 5.0
ROI area for T8T9 (mm²)	10.0 ± 4.2	10.3 ± 3.6
ROI area for T11T12 (mm²)	10.6 ± 2.3	10.9 ± 4.3
ROI area for L1L2 (mm²)	10.8 ± 4.1	12.9 ± 3.2
ICC: ROI area	0.899	0.901
ICC: Peak diastolic flow	0.953	0.944
ICC: Peak systolic flow	0.977	0.944

CSF Flow:

Bidirectional (cranial-caudal) CSF flow across the cardiac cycle was observed at all spinal levels (Fig. 2). At the thoracolumbar levels, the net flow waveform had similar shape and amplitude for all animals. The standard deviation at C2/C3 across the cardiac cycle was larger than at the other spinal levels.

The effect of spinal level on peak systolic flow was dependent on SAS region (p < 0.001) (Fig. 3). In the dorsal SAS, it was greater at C2/C3 (dorsal: -0.36 ± 0.15) compared to the thoracolumbar levels: T8/T9 (-0.04 ± 0.03 mL/s) (estimate = -0.34, 95% CI [-0.43, -0.24], p < 0.001), T11/T12 (-0.07 ± 0.06 mL/s) (estimate = -0.30, 95% CI [ -0.39, -0.21], p < 0.001), L1/L2 (-0.07 ± 0.05 mL/s) (estimate = -0.30, 95% CI [-0.38, -0.21, p < 0.001). In the ventral SAS, peak flow was also greater at C2/C3 (-0.18 ± 0.5 mL/s) compared to T8/T9 (-0.08 ± 0.08 mL/s) (estimate = -0.09, 95% CI [-0.18, -0.00], p = 0.046), but there was no difference between C2/C3 and T11/T12 (-0.08 ± 0.08 mL/s) (estimate = -0.08, 95% CI [-0.15, -0.01], p = 0.128) and C2/C3 and L1/L2 (-0.09 ± 0.06 mL/s) (estimate = -0.08, 95% CI [-0.17, -0.01], p = 0.126). In both SAS regions there were no differences between T8/T9 and T11/T12 (dorsal: estimate = 0.04, 95% CI [-0.06, 0.14], p = 1.000; ventral: estimate = 0.01, 95% CI [-0.08, 0.10], p = 1.000;), and T11/T12 and L1/L2 (dorsal: estimate = 0.00, 95% CI [-0.09, 0.09], p = 1.000; ventral: estimate = 0.01, 95% CI [-0.08, 0.09], p = 1.000).

The effect of spinal level on peak diastolic flow was dependent on SAS region (p < 0.001). Peak diastolic flow in the dorsal SAS was greater at C2/C3 (0.16 ± 0.08 mL/s) than the thoracolumbar levels: T8/T9 (0.03 ± 0.02 mL/s) (estimate = 0.14, 95% CI [0.08, 0.20], p < 0.001), T11/T12 (dorsal: 0.05 ± 0.02 mL/s; estimate = 0.13, 95% CI [0.07, 0.18], p < 0.001), L1/L2 (dorsal: 0.04 ± 0.04 mL/s; estimate = 0.12, 95% CI [0.06, 0.17], p < 0.001). In the ventral SAS, there was no difference in peak diastolic flow between C2/C3 (0.07 ± 0.06 mL/s) and the thoracolumbar levels: T8/T9 (0.08 ± 0.08 mL/s; estimate = -0.02, 95% CI [-0.08, 0.04], p = 1.000), T11/T12 (0.07 ± 0.08 mL/s; estimate = -0.02, 95% CI [-0.08, 0.05], p = 1.000), and L1/L2 (0.08 ± 0.06 mL/s; estimate = -0.03, 95% CI [-0.08, 0.03], p = 1.000). There were no differences in both SAS regions, between T8/T9 and T11/T12 (dorsal: estimate = -0.02, 95% CI [-0.08, 0.05], p = 1.000; ventral: estimate = 0.00, 95% CI [-0.06, 0.06], p = 1.000), and T11/T12 and L1/L2 (dorsal: estimate = -0.01, 95% CI [-0.07, 0.05], p = 1.000; ventral: estimate = -0.01, 95% CI [-0.07, 0.05], p = 1.000). Estimated marginal means are available in Additional file 5: S4, and complete pairwise comparisons are available in Additional file 6: Table S4.

Maximum CSF Velocity:

The effect of spinal level on maximum CSF velocity in the caudal direction was dependent on SAS region (p = 0.017) (Fig. 4). In the dorsal SAS, maximum CSF velocity in the caudal direction was greater at C2/C3 (-7.50 ± 2.02 cm/s) than each of the thoracolumbar levels: T8/T9 (-1.49 ± 1.11; estimate = -6.29, 95% CI [-8.24, -4.34], p < 0.001), T11/T12 (-2.78 ± 1.43; estimate = -4.81, 95% CI [-6.67, -2.94], p < 0.001), L1/L2 (-2.31 ± 1.63; estimate = -5.24, 95% CI [-6.98, -3.50], p < 0.001). In the ventral SAS, maximum CSF velocity in the caudal direction was also greater at C2/C3 (-5.82 ± 2.80 cm/s) than the thoracolumbar levels: T8/T9 (-2.27 ± 1.18 cm/s; estimate = -3.34, 95% CI [-5.23, -1.45], p < 0.001), T11/T12 (-3.32 ± 1.39 cm/s; estimate = -2.11, 95% CI [-4.12, -0.94], p = 0.035), and L1/L2 (-2.32 ± 1.81 cm/s; estimate = -3.29, 95% CI [-5.15, -1.43], p < 0.001). There was no difference between T8/T9 and T11/T12 (dorsal: estimate = 1.48, 95% CI [-0.59, 3.56], p = 0.334; ventral: estimate = 1.23, 95% CI [-0.66, 3.13], p = 0.483), and T11/T12 and L1/L2 (dorsal: estimate = -0.43, 95% CI [-2.34, 1.48)], p = 1.000; ventral: -1.18, 95% CI [-3.04, 0.68], p = 0.531) in both SAS regions.

The effect of spinal level on maximum CSF velocity in the cranial direction was dependent on SAS region (p = 0.001). In the dorsal SAS, maximum CSF velocity in the cranial direction was greater at C2/C3 (dorsal: 6.63 ± 2.12 cm/s, ventral: 3.84 ± 2.0 cm/s) than the thoracolumbar levels: T8/T9 (1.76 ± 1.09 cm/s; estimate = 5.12, 95% CI [3.33, 6.90], p < 0.001), T11/T12 (2.72 ± 1.39 cm/s; estimate = 3.91, 95% CI [2.20, 5.16], p < 0.001), L1/L2 (2.51 ± 1.53 cm/s; estimate = 4.14, 95% CI [2.55, 5.73], p < 0.001). In the ventral SAS, there were no differences in maximum cranial velocity between C2/C3 and the thoracolumbar levels: T8/T9 (2.0 ± 1.32 cm/s; estimate = 1.62, 95% CI [-0.11, 3.36], p = 0.079), T11/T12 (2.44 ± 1.26 cm/s; estimate = 0.99, 95% CI [-0.85, 2.83], p = 0.886), and L1/L2 (2.02 ± 1.50 cm/s; estimate = 1.63, 95% CI [-0.08, 3.33], p = 0.070). There were no differences between T8/T9 and T11/T12 (dorsal: estimate = -1.21, 95% [-3.11, 0.69], p = 0.527; ventral: estimate = -0.63, 95% CI [-2.37, 1.100], p = 1.000), and T11/T12 and L1/L2 (dorsal: estimate = 0.23, 95% [-1.51, 1.98], p = 1.000; ventral: estimate = 0.64, 95% CI [-1.07, 2.34], p = 1.000) in both regions. Estimated marginal means are available in Additional file 5: S4, and complete pairwise comparisons are available in Additional file 6: Table S4.

Velocity Wave Propagation Speed:

The mean CSF velocity waveforms (Additional file 7: Figure S3) demonstrated a phase shift from C2/C3 to L1/L2, indicating propagation of the velocity wave in the caudal direction in both the dorsal and ventral regions (Fig. 5A-B). The mean distance between C2/C3 and L1/L2 was 416 ± 24 mm. Peak systolic velocity at C2/C3 was close to coincident with peak diastolic CSF velocity at T8/T9, T11/T12 and L1/L2 (Additional file 8: Table S5). Peak systolic velocity at C2/C3 occurred at 70.0 ± 6.3% (392.9 ± 35.5 ms) of the cardiac cycle in the dorsal SAS and at 64.8 ± 9.3% (341.1 ± 49.1 ms) of the cardiac cycle in the ventral SAS. Peak diastolic velocity at L1/L2 occurred at 74.9 ± 10.3% (391.3 ± 54.0 ms ) of the cardiac cycle in the dorsal SAS, and at 80.0 ± 7.4% (405.4 ± 37.5) in the ventral SAS. Systolic (caudal) VWS (C2/C3 to L1/L2) was 1.02 ± 0.25 m/s in the dorsal SAS and 0.91 ± 0.22 m/s in the ventral SAS, and diastolic (cranial) VWS was 1.41 ± 0.39 m/s in the dorsal SAS and 1.22 ± 0.21 m/s in the ventral SAS (Fig. 5).

CSF dynamics along the spine have only recently been characterised in humans and NHP (macaca fascicularis) (15–19). Large animal models are increasingly being used to investigate SCI (27, 31). The current study provides normative data for spinal CSF flow in experimentally naïve anaesthetised domestic pigs as a baseline and comparison for future SCI studies. The study provides evidence that spinal CSF flow in anaesthetised and ventilated pigs is lower, and has slower velocity wave propagation, than in conscious humans, but is similar to that in anaesthetised NHP (15, 19, 34, 36).

Spinal net CSF flow in anaesthetised pigs is considerably lower than in conscious humans across the cardiac cycle. Peak systolic CSF flow measured in the cervical (C4; 2.836 ± 0.238 mL/s) and lumbar (L1, L2 or L3; 1.260 ± 0.213 mL/s ) spine of awake humans is approximately eight times greater at both levels than in the anaesthetised pigs (15). Peak diastolic flow in the cervical spine is approximately fifteen times (C4; 1.486 ± 0.198 mL/s) greater, and in the lumbar spine is approximately three times (L1, L2 or L3; 0.295 ± 0.121 mL/s) greater, in humans than in the current pig study (15). In healthy conscious humans, peak mean velocity in the lower thoracic region (5.81 ± 1.42 cm/s; N = 14) (37) is greater than in the anaesthetised pig (Additional file 10: Table S6). The SAS is also larger in humans than the pig: for example, the T10 anterior-posterior diameter of the CSF (dural minus spinal cord diameter) in the domestic pig is 2.03 mm [1.28 mm – 3.11 mm] (median and range; ultrasound measurements) (38), whereas in humans it is 7.4 ± 3.1 mm (mean ± SD; computed tomography contrast myelogram measurements) (39). Together, these results suggest that higher CSF flow in conscious humans than anaesthetised pigs results, at least in part, from the combined effect of a larger SAS and greater CSF velocity in humans. CSF flow in the pig was similar to that reported for anaesthetised, but not mechanically ventilated, NHPs: peak systolic flow in the cervical spine was approximately 1.5 times greater in the NHP (0.5 ± 0.2 mL/s, C2/C3; estimated from graphical data), and in the lumbar spine was slightly lower (0.1 ± 0.05 mL/s, L3/L4; estimated from graphical data), than in the pigs. NHP cervical peak diastolic flow was nearly two times smaller than (0.2 ± 0.2 mL/s; estimated from graphical data), and lumbar flow was approximately the same as (0.1 ± 0.1 mL/s; estimated from graphical data), that in the current pig study (19). In pigs, peak systolic CSF flow decreased from the cervical to the thoracic levels, but was unchanged at the thoracolumbar levels both dorsally and ventrally. In humans, peak systolic and diastolic flow decreases caudally to a minimum in the lumbar region (15, 18). Such a reduction across the spine may not have been observed in the current study because the thoracic (T8/T9) and lumbar (L1/L2) measurements encompassed a limited span (193 ± 20 mm). It is also possible that respiratory forces have a greater effect on CSF flow in the thoracic region compared to at the lumbar levels (16), and that no difference was observed between adjacent thoracic and lumbar sites due to mechanical ventilation of the pigs.

Although the relative contribution of respiratory and cardiac cycles to CSF dynamics is unclear, it is evident that physiological variations of both can alter CSF flow (40–43). It is commonly thought that CSF travels cranially with inhalation, and caudally with exhalation, due to changes in intrathoracic pressure with spontaneous breathing (16, 17, 41, 42). Unlike in most, non-acute, clinical CSF flow studies reported, the animals in this study were anesthetised and ventilated, which causes positive intrathoracic pressure and produces increased pressure during inhalation and decreased pressure during exhalation (the opposite of spontaneous breathing). Positive intrathoracic pressure throughout the respiratory cycle may contribute to lower CSF flow. Mechanically ventilated rats had less movement of fluorescent tracer in the spinal SAS in the caudal direction compared to spontaneously breathing animals (40). This suggests that respiratory conditions need to be carefully considered in CSF flow study comparisons. Mechanical ventilation can also influence arterial pulse pressure through complex cardiopulmonary interactions (44). It is apparent that reduced arterial pulse pressure decreases CSF flow in the spinal perivascular spaces (45, 46), but its effect on CSF pulsations in the SAS is unknown. There is limited and conflicting evidence that heart rate influences CSF flow: increasing heart rate resulted in increased bidirectional CSF flow velocities in a three dimensional computational fluid dynamics model of the SAS (43), while a study in rats showed that increased heart rate had little influence on CSF flow (40). The heart rate range in this study (71–174 bpm) is larger than in the other animal studies (canines, 70 − 110 bpm; NHP, 92–132 bpm). In this current study, two animals had notably higher heart rates (P004, 120–130 bpm; P012, 174 bpm), both with apparent abnormal cardiac gating. The animal (P006) with markedly higher peak systolic and diastolic CSF flow across most spinal levels in the ventral region (Additional file 9: Table S7) had unremarkable physiological parameters (Additional file 4: Table S2). Anaesthesia may also contribute to CSF dynamics (47), and this is potentially due to its influence on blood pressure and partial pressure of carbon dioxide in the blood stream. Mean arterial pressure is thought to influence CSF flow in the cerebral perivascular spaces (48); however, its effect on pulsating spinal CSF flow in the SAS is understudied. In one rat study there was no effect of mean arterial pressure on spinal SAS CSF flow (40). In the current study, the animal that received a propofol bolus during the scan (P014; T11/T12 and L1/L2 scanned prior to bolus, and T8/T9 and C2/C3 after bolus) had abnormally higher peak systolic flow at T11/T12 and L1/L2 dorsally, T8/T9 and L1/L2 ventrally and peak diastolic flow at T8/T9 dorsally. The propofol bolus was indicated because the animal was not deeply anaesthetised (as evidenced by physical movement), and was therefore likely to have higher blood pressure; however, blood pressure was not measured concurrently in this study. In addition, cerebral blood flow is influenced by the partial pressure of arterial carbon dioxide (49). Hypercapnia increases cerebral blood flow and intracranial pressure (50), which may effect spinal CSF flow due to the potential interaction between intracerebral arteries and pulse propagation (51). It is becoming increasingly clear that respiratory forces have a large influence on CSF flow; however, the potential contributions of other physiological parameters such as heart rate, blood pressure, and carbon dioxide in the bloodstream, need to be further investigated to establish their relative effect on CSF dynamics. These physiological parameters should be measured and recorded during PC-MRI acquisition of CSF flow in future studies.

Velocity wave propagation was detected in the caudal direction along the spinal axis in laterally recumbent pigs. Velocity wave propagation speed can be used to approximate pulse wave velocity (pressure wave propagation) since it has been shown that they are nearly identical under certain conditions (36). Because of this, some studies use the term ‘pulse wave velocity’ rather than VWS, for the identical measure(19, 34). It has been hypothesised that CSF pressure waves originate in the intracerebral arteries and propagate in a caudal direction (51); however, the origin of the pulse remains unresolved since there are other studies which suggest local sources (15, 52, 53). In cardiovascular diagnostics, pulse wave velocity is a measure of vessel compliance. VWS has recently been reported for spinal CSF flow studies (34, 36, 54) where it likely reflects compliance of the spinal cord tissue, dura, and surrounding tissues (55). A one-dimensional tube model of the spinal SAS has been used to show that increasing spinal compliance results in slower VWS and greater attenuation of the pulse (56). In healthy conscious humans, VWS is three (4.6 ± 1.7 m/s)(36) to four (5.83 ± 3.36 m/s) (34) times faster than that measured in the pigs. In NHP, VWS is similar to the VWS estimated for the dorsal SAS in this study (1.13 m/s) (19). In the current study, VWS was calculated between C2/C3 and L1/L2, rather than between each adjacent spinal levels. Because the lower spinal levels sampled were concentrated around T10, the increased distance, and therefore pulse transit times, between these locations should improve temporal accuracy but remove detection of region-specific VWS. Further study is necessary to elucidate the relevance of VWS along the spine, and locally, in healthy and diseased states.

Defining dorsal and ventral regions in the SAS is likely to be beneficial for future CSF flow investigations in the context of SCI, where SAS occlusion may not be uniformly distributed. The current study suggests that, in these pigs, CSF flow and maximum velocity were dependent on SAS region at C2/C3. While the majority of studies do not report CSF flow in separate SAS regions, a study using dynamic sagittal PC-MRI scans on cervical myelopathy patients found that grade 2 cervical stenosis was more frequently associated with interrupted flow patterns in the ventral or dorsal SAS (20). A study of Chiari malformation in canines reported that syringomyelia was associated with lower peak velocity in the dorsal SAS (foramen magnum and C2/C3) but not in the ventral SAS (57). Together, these observations suggest the potential importance of considering local CSF flow characteristics. In the current study, CSF flow was generally similar, but not identical, in the two regions, and at spinal level T8/T9, four animals had no flow signal in the dorsal SAS while CSF flow was observed in the ventral SAS. There is some evidence that CSF velocity is higher ventrally during both systolic and diastolic flow, in the human cervical SAS (58); therefore, it is possible that in these animals flow in the dorsal SAS was not detected by PC-MRI because of lower velocity. In addition, the variability of maximum CSF velocity between animals in this study suggests that multi-V_ENC scans should be run in the thoracolumbar spine. Maximum CSF velocity at C2/C3 also exceeded the applied V_ENC (6 cm/s) in 75% of the scans in the first ten animals. Aliasing can be difficult to detect prior to image post-processing, therefore “real-time” post-processing should be completed immediately after the scan to allow for immediate re-scanning as needed. A V_ENC of at least 8 cm/s at C2/C3 is recommended to minimise the risk of aliasing, in pigs.

There are several limitations in this study. The sample size was limited; however, the number of animals used in this study is greater than that reported for similar animal studies characterising CSF flow in NHP (macaca fascicularis; N = 8) (19), and quantifying CSF velocity in canines (beagle, N = 6) (59). In addition, the age and sex of the animals in this study were selected because of their relevance to the associated SCI experiments; it is not known if CSF flow characteristics change with animal age, size and sex. The spinal levels selected were of particular relevance to pig SCI contusion models, and may not be generalisable to levels of interest for the study of other spinal pathologies. Blood pressure was not measured concurrently with image acquisition, so blood pressure could not be identified as a physiological contributor to variability in CSF flow. The cause of the apparent abnormal cardiac gating in two animals could not be identified; the pulse oximeter signal was not acquired for post-processing. In addition, the cause of undetected CSF flow in three animals could not be identified; however, technical failures and/or insufficient CSF flow have also been reported in human PC-MRI studies (15, 17, 34, 54).

This study demonstrates that CSF flow in the pig can be detected and quantified with PC-MRI using a 3T MR scanner. Spinal CSF flow in the healthy, anaesthetised, ventilated domestic pig in lateral recumbency is lower, and has a lower velocity wave propagation speed, than in conscious humans. These data are a normative baseline at spinal levels relevant for future SCI research in this animal model, and provide a means to validate computational models of the pig SAS.

CI: Confidence interval

CNS: Central nervous system

CSF: Cerebrospinal fluid

C2/C3: Cervical spine between levels 2 and 3

LMM: Linear mixed effects model

L1/L2: Lumbar spine between levels 1 and 2

MRI: Magnetic resonance imaging

NHP: Non-human primates

PC-MRI: Phase contract magnetic resonance imaging

ROI: Region of interest

SAS: Subarachnoid space

SCI: Spinal cord injury

SD: Standard deviation

T8/T9: Thoracic spine between levels 8 and 9

T11/T12: Thoracic spine between levels 11 and 12

VWS: Velocity wave speed

Ethics approval and consent to participate:

Consent for publication

Not applicable

Availability of data and materials

The datasets generated and/or analysed during the current study are available in the Figshare repository, 10.25909/20494356

Competing interests

The authors declare that they have no competing interests

Funding

This study was supported by a Basic Research Grant Award from the North American Spine Society (CFJ, AVL). MAB is supported by an Australian Government Research Training Program Scholarship (RTPS). CDG is supported by a The Hospital Research Foundation PhD Scholarship. RDQ is supported by an Australian Research Council Discovery Project (DP190101209). VK received support from the Swiss National Science Foundation (205321_182683). CFJ was partially supported by a National Health and Medical Research Council (Australia) Early Career Fellowship during this study (APP1072387).

Authors’ contributions

Conceptualization, MAB, AVL, and CFJ; Methodology, MAB, CDG, AW, VK, and CFJ; Validation, MAB, AW, VK and CFJ; Formal Analysis, MAB, RDQ, and CFJ; Investigation, MAB, CDG, and CFJ; Resources, MAB and CFJ; Data Curation, MAB, CDG, and CFJ; Writing – Original Draft, MAB and CDF; Writing – Review & Editing, MAB, CDG, RDQ, AW, AVL, VK, and CFJ; Visualization, MAB, RDQ, and CFJ; Supervision, AVL and CFJ; Project Administration, MAB, CDG, AVL, and CFJ; Funding Acquisition, AVL and CFJ.

Acknowledgments

The authors acknowledge the facilities, and scientific and technical assistance, of the National Imaging Facility, a National Collaborative Research Infrastructure (NCRIS) capability, at the Large Animal Research and Imaging Facility (LARIF), South Australian Health and Medical Research Institute (SAHMRI).

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No competing interests reported.

Additionalfile1.bmp
Additional file 1: Figure S1 (.bmp)Exemplar PC-MRI (A) magnitude (B) phase images at peak diastolic flow with two ROIs in the dorsal SAS dependent on flow signal within that region (P009). This was to ensure that the included pixels within the ROI reflect a CSF flow signal, and to avoid regions with no flow signal. PC-MRI = phase contrast magnetic resonance imaging
Additionalfile2.xlsx
Additional file 2: Table S1 (.xlsx)The number of ROI and area of ROI (mm²) drawn in the dorsal or ventral SAS dependent on detectable CSF flow signal within that region, where 0 corresponds to no CSF flow detected in that region. (*) animals excluded from temporal data analyses and (**) animals excluded from all analyses. ROI = region of interest, CSF = cerebrospinal fluid.
Additionalfile3.bmp
Additional file 3: Figure S2 (.bmp)Experimental flow data from P004 and P012 were excluded from temporal analyses and plots due to abnormal cardiac gating. Animal P004 in the dorsal (A) and ventral (B) SAS: the full CSF flow oscillation (diastolic and systolic flow) was not captured in the cycle. Animal P012 in the dorsal (C) and ventral (D) SAS: systolic pulse was acquired at the beginning of the cycle instead of the diastolic pulse. These temporal errors are probably due to incorrect cardiac gating. SAS = subarachnoid space.
Additionalfile4.xlsx
Additional file 4: Table S2 (.xlsx)Physiological recordings and anaesthetic drug levels of each animal during the PC-MRI scan. (*) animals excluded from temporal data analyses, and (**) animals excluded from all analyses. PC-MRI = phase contrast magnetic resonance imaging.
Additionalfile5.xlsx
Additional file 5: Table S3 (.xlsx)Estimated marginal means with 95% CI for peak diastolic and systolic flow, and maximum cranial and caudal velocity from LMMs. LMM = linear mixed effects models
Additionalfile6.xlsx
Additional file 6: Table S4 (.xlsx)Summary of results from the LMM pairwise comparisons for peak systolic and diastolic flow and maximum cranial and caudal flow. There was a significant interaction factor between spinal level and SAS region for all four outcome measures. LMM = linear mixed effects models
Additionalfile7.bmp
Additional file 7: Figure S3 (.bmp)Mean CSF velocity in the dorsal and ventral region of the SAS at each spinal level. Data presented as means ± one standard deviation as shaded area. CSF = cerebrospinal fluid, SAS = subarachnoid space.
Additionalfile8.xlsx
Additional file 8: Table S5 (.xlsx)Time to peak systolic and diastolic velocity (as a percentage of the cardiac cycle) and cardiac cycle duration (ms) for each spinal level and SAS region. SAS = subarachnoid space.
Additionalfile9.xlsx
Additional file 9: Table S6 (.xlsx)Peak mean systolic and diastolic velocity values (mL/s) for each spinal level and SAS region, with mean ± one standard deviation. SAS = subarachnoid space.
Additionalfile10.xlsx
Additional file 10: Table S7 (.xlsx)Peak systolic and diastolic flow values (mL/s) for each spinal level and SAS region, with mean ± one standard deviation. SAS = subarachnoid space.

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Characterising spinal cerebrospinal fluid flow in the pig with phase-contrast magnetic resonance imaging

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Animal Ethics:

Animals:

Anaesthetic Protocol:

Magnetic Resonance Imaging:

Phase Contrast Magnetic Resonance Imaging Acquisition:

Phase Contrast Magnetic Resonance Imaging Post-Processing:

Velocity Wave Propagation Speed

Statistical Analyses:

Results

CSF Flow:

Maximum CSF Velocity:

Velocity Wave Propagation Speed:

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1