Spatial and Diurnal Variations in Sodium Content Within Intervertebral Disc Tissue

Abstract

Background: Daily cycles in tissue osmolarity within the intervertebral disc (IVD) are a biophysical signal that regulates cellular metabolism and arise from deformation of the charged and hydrated extracellular matrix (ECM). However, the magnitude of these osmotic cycles remains unclear due to spatial variations in ECM composition and fixed charge density (FCD) between the regions of the IVD and between the pericellular matrix (PCM) and surrounding ECM. This study aimed to (1) validate the use of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to assess intra-tissue sodium content and (2) apply this method to assess temporal and spatial changes in intra-tissue sodium content during a diurnal loading cycle.

Methods: The FCD of nucleus pulposus (NP) tissue was modified via equilibrium dialysis, and intra-tissue sodium was assessed via inductively coupled plasma-optical emission spectroscopy (ICP-OES) and LA-ICP-MS. In addition, tissue was stained with a cationic iodine-based contrast agent, and iodine was assessed via LA-ICP-MS. Diurnal changes in sodium were measured via LA-ICP-MS and ICP-OES in bovine caudal motion segments loaded under simulated physiologic loading for 40 (n = 4) or 48 (n = 4) hours, representing end-of-day and end-of-night deformations, respectively.

Results: Intra-tissue sodium content assessed via LA-ICP-MS strongly correlated (r ≥ 0.95) with ICP-OES sodium content, theoretical FCD values, and LA-ICP-MS iodine content in equilibrated tissue. Diurnal sodium results demonstrate that at the macroscale the NP region possessed a greater sodium content than the annulus fibrosus (AF) and experienced greater diurnal changes ("end-of-day-NP" sodium [0.52 ± 0.18 mEq Na/g wet wt], "end of night-NP" sodium [0.34 ± 0.13 mEq Na/g wet wt]) than the AF which did not experience a diurnal change. At the microscale, average PCM sodium normalized to adjacent ECM sodium at the end-of-day (2.3 ± 0.96) was greater than at the end-of-night (1.5 ± 0.38), indicating cells embedded within their specialized PCM likely experience elevated osmotic fluctuations.

Keywords: fixed charge density; laser ablation‐inductively coupled plasma‐mass spectrometry; pericellular matrix; tissue sodium.

FIGURE 1

Measurements of intra‐tissue sodium and iodine with increasing osmotic pressure: Results demonstrate LA‐ICP‐MS measurements of sodium and iodine content correlate with ICP‐OES measurements of sodium and therefore LA‐ICP‐MS measurements can give spatial measurements of fixed charge density (FCD). (A) Schematic of study design where after bovine nucleus pulposus tissue was equilibrated in 5%, 10%, 20%, or 25% PEG in 0.15 M a tissue plug was taken for: (1) measurement of sodium content via inductively coupled plasma‐optical emission spectroscopy (ICP‐OES), or frozen and cryo‐sectioned for measures of sodium (2) or stained with an anionic iodine contrast agent (3) and each element measured via laser ablation‐inductively coupled plasma‐mass spectrometry (LA‐ICP‐MS). (B) Schematic of the LA‐ICP‐MS process in which 3 lines of 10 ablations, each 100 μm², are ablated from sectioned tissue. The area under the curve was quantified. (C) Measurements of (top) sodium content and theoretical fixed charge density (FCD) via ICP‐OES normalized to total tissue water, (middle) sodium content via LA‐ICP‐MS, and (bottom) iodine content via LA‐ICP‐MS versus osmotic pressure demonstrating an increase in sodium content/theoretical fixed charge density with increasing osmotic pressure. (D) Correlation of ICP‐OES sodium concentration and theoretical FCD with LA‐ICP‐MS measurements of sodium content in PEG equilibrated tissue. (E) Correlation of ICP‐OES sodium concentration and theoretical FCD with LA‐ICP‐MS iodine content. (F) Correlation between LA‐ICP‐MS sodium and LA‐ICP‐MS iodine measurements.

FIGURE 2

Diurnal loading protocol: (A) Applied mechanical stress and representative resulting strain applied to motion segments over a 40 or 48 h period (N = 4/timepoint). (B) Change in average total strain and (C) strain relative to 24 h. (D) Percent water content and (E) tissue hydration in the nucleus pulposus (NP) and annulus fibrosus (AF) regions. *p < 0.05 for a t‐test between time points within region and the p values from a two‐way ANOVA are shown in the plot. N = 4/region/time.

FIGURE 3

(A) Schematic of tissue preparation for ICP‐OES and LA‐ICP‐MS in the nucleus pulposus (NP) and annulus fibrosus (AF) regions. Measurements of sodium concentration normalized to tissue hydration via (B) ICP‐OES and (C) LA‐ICP‐MS in NP and AF tissue after diurnal testing. Each circle is the average of the 30 ablation regions, *p < 0.05 for a t‐test between time points within region and the p values from a two‐way ANOVA are shown in the plot. N = 4/region/time.

FIGURE 4

Correlation of intra‐tissue sodium of annulus fibrosus (AF) and nucleus pulposus (NP) tissue calculated via ICP‐OES and LA‐ICP‐MS.

FIGURE 5

(A) Schematic of tissue preparation for laser ablation‐inductively coupled plasma‐mass spectrometry (LA‐ICP‐MS) in the extracellular (ECM) and pericellular matrix (PCM) of nucleus pulposus (NP) cells. (B) The PCM to ECM ratio of sodium content from a cell and adjacent region at end‐of‐day (n = 15 cells from 3 discs) and end‐of‐night (n = 20 cells from 2 discs). *p < 0.05 for a t‐test between time points within region.