Practical implementation of the planar and spatial rotator in a complex tissue: the brain.

In neuroscience, application of widely used stereological local volume estimators, including the planar rotator, is challenged by the combination of a complex tissue organisation and an estimator requirement of either isotropic or vertical sections, i.e. randomly oriented tissue. The spatial rotator is applicable with any tissue orientation but is sensitive to projection artefacts. The challenge is thus to select the most appropriate method for individual analyses. In this study, agreement between estimates of mean cell volume acquired with the vertical planar and the spatial rotator is assessed for two brain regions with different types of cytoarchitecture (motor cortex and hippocampal cornu ammonis 1). The possibility of using the planar rotator in tissues cut in an arbitrary direction is explored and requirements for a theoretically unbiased result as well as histological considerations are provided. LAY DESCRIPTION: Cells may change volume both during disease and with advancing age. Assessment of the volume of individual cells can therefore serve as a useful indicator of general tissue state. Most available methods to estimate cell volume in tissue sections, however, require that the tissue analysed has random orientation. Particularly for complex tissues such as the brain this is a challenge as identification, delineation and subdivision of many brain areas rely heavily on the use of anatomical atlases where illustrations depict the tissue in a few well-known orientations. In this study, the practical application of two different methods for estimating mean cell volumes in tissues cut in a preferred orientation is evaluated. Requirements for the feasibility of cell volume estimation without random tissue orientation as well as histological considerations are provided.

IUGR decreases cardiomyocyte endowment and alters cardiac metabolism in a sex- and cause-of-IUGR-specific manner.

Intrauterine growth restriction (IUGR) increases the risk of ischemic heart disease in adulthood. Studies in rats suggest cardiac vulnerability is more pronounced in males and in offspring that were exposed to hypoxia in utero. Therefore, we aimed to test the hypotheses that 1) IUGR adolescent males, but not females, have fewer cardiomyocytes and altered expression of cardiometabolic genes compared with controls; and 2) IUGR due to hypoxia has a greater effect on these parameters compared with IUGR due to nutrient restriction. IUGR was induced in guinea pigs by maternal hypoxia (MH; 10% O2, n = 9) or maternal nutrient restriction (MNR; ~30% reduction in food intake, n = 9) in the second half of pregnancy and compared with control ( n = 11). At 120 days of age, postmortem was performed and the left ventricle perfusion fixed for stereological determination of cardiomyocyte number or snap frozen to determine the abundance of cardiometabolic genes and proteins by quantitative RT-PCR and Western blotting, respectively. MH reduced the number of cardiomyocytes in female ( P < 0.05), but not male or MNR, adolescent offspring. Furthermore, IUGR males had decreased expression of genes responsible for fatty acid activation in the sarcoplasm ( FACS) and transport into the mitochondria ( AMPK-a2 and ACC; P < 0.05) and females exposed to MH had increased activation/phosphorylation of AMP-activated protein kinase-α ( P < 0.05). We postulate that the changes in cardiomyocyte endowment and cardiac gene expression observed in the present study are a direct result of in utero programming, as offspring at this age did not suffer from obesity, hypertension, or left ventricular hypertrophy.

SorCS2 is required for BDNF-dependent plasticity in the hippocampus.

SorCS2 is a member of the Vps10p-domain receptor gene family receptors with critical roles in the control of neuronal viability and function. Several genetic studies have suggested SORCS2 to confer risk of bipolar disorder, schizophrenia and attention deficit-hyperactivity disorder. Here we report that hippocampal N-methyl-d-aspartate receptor-dependent synaptic plasticity is eliminated in SorCS2-deficient mice. This defect was traced to the ability of SorCS2 to form complexes with the neurotrophin receptor p75NTR, required for pro-brain-derived neurotrophic factor (BDNF) to induce long-term depression, and with the BDNF receptor tyrosine kinase TrkB to elicit long-term potentiation. Although the interaction with p75NTR was static, SorCS2 bound to TrkB in an activity-dependent manner to facilitate its translocation to postsynaptic densities for synaptic tagging and maintenance of synaptic potentiation. Neurons lacking SorCS2 failed to respond to BDNF by TrkB autophosphorylation, and activation of downstream signaling cascades, impacting neurite outgrowth and spine formation. Accordingly, Sorcs2-/- mice displayed impaired formation of long-term memory, increased risk taking and stimulus seeking behavior, enhanced susceptibility to stress and impaired prepulse inhibition. Our results identify SorCS2 as an indispensable coreceptor for p75NTR and TrkB in hippocampal neurons and suggest SORCS2 as the link between proBDNF/BDNF signaling and mental disorders.

Improved sampling and analysis of images in corneal confocal microscopy.

INTRODUCTION: Corneal confocal microscopy (CCM) is a noninvasive clinical method to analyse and quantify corneal nerve fibres in vivo. Although the CCM technique is in constant progress, there are methodological limitations in terms of sampling of images and objectivity of the nerve quantification. The aim of this study was to present a randomized sampling method of the CCM images and to develop an adjusted area-dependent image analysis. Furthermore, a manual nerve fibre analysis method was compared to a fully automated method. METHODS: 23 idiopathic small-fibre neuropathy patients were investigated using CCM. Corneal nerve fibre length density (CNFL) and corneal nerve fibre branch density (CNBD) were determined in both a manual and automatic manner. Differences in CNFL and CNBD between (1) the randomized and the most common sampling method, (2) the adjusted and the unadjusted area and (3) the manual and automated quantification method were investigated. RESULTS: The CNFL values were significantly lower when using the randomized sampling method compared to the most common method (p = 0.01). There was not a statistical significant difference in the CNBD values between the randomized and the most common sampling method (p = 0.85). CNFL and CNBD values were increased when using the adjusted area compared to the standard area. Additionally, the study found a significant increase in the CNFL and CNBD values when using the manual method compared to the automatic method (p ≤ 0.001). CONCLUSION: The study demonstrated a significant difference in the CNFL values between the randomized and common sampling method indicating the importance of clear guidelines for the image sampling. The increase in CNFL and CNBD values when using the adjusted cornea area is not surprising. The observed increases in both CNFL and CNBD values when using the manual method of nerve quantification compared to the automatic method are consistent with earlier findings. This study underlines the importance of improving the analysis of the CCM images in order to obtain more objective corneal nerve fibre measurements.

3D analysis of synaptic vesicle density and distribution after acute foot-shock stress by using serial section transmission electron microscopy.

Behavioural stress has shown to strongly affect neurotransmission within the neocortex. In this study, we analysed the effect of an acute stress model on density and distribution of neurotransmitter-containing vesicles within medial prefrontal cortex. Serial section transmission electron microscopy was employed to compare two groups of male rats: (1) rats subjected to foot-shock stress and (2) rats with sham stress as control group. Two-dimensional (2D) density measures are common in microscopic images and are estimated by following a 2D path in-section. However, this method ignores the slant of the active zone and thickness of the section. In fact, the active zone is a surface in three-dimension (3D) and the 2D measures do not accurately reflect the geometric configuration unless the active zone is perpendicular to the sectioning angle. We investigated synaptic vesicle density as a function of distance from the active zone in 3D. We reconstructed a 3D dataset by estimating the thickness of all sections and by registering all the image sections into a common coordinate system. Finally, we estimated the density as the average number of vesicles per area and volume and modelled the synaptic vesicle distribution by fitting a one-dimensional parametrized distribution that took into account the location uncertainty due to section thickness. Our results showed a clear structural difference in synaptic vesicle density and distribution between stressed and control group with improved separation by 3D measures in comparison to the 2D measures. Our results showed that acute foot-shock stress exposure significantly affected both the spatial distribution and density of the synaptic vesicles within the presynaptic terminal.

Fast diffusion kurtosis imaging of fibrotic mouse kidneys.

Diffusion kurtosis imaging (DKI) is sensitive to tissue microstructure and may therefore be useful in the diagnosis and monitoring of disease in brain and body organs. Generally, diffusion magnetic resonance imaging (dMRI) in the body is challenging because of the heterogeneous body composition, which can cause image artefacts as a result of chemical shifts and susceptibility differences. In addition, the abdomen possesses physiological factors (e.g. breathing, heartbeat, blood flow) which may severely reduce image quality, especially when echo planar imaging is employed, as is typical in dMRI. Collectively, these challenging measurement conditions impede the use and exploration of DKI in the body. This impediment is further exacerbated by the traditionally large amount of data required for DKI and the low signal-to-noise ratio at the b-values needed to effectively probe the kurtosis regime. Recently introduced fast DKI techniques reduce the challenge of DKI in the body by decreasing the data requirement substantially, so that, for example, triggering and breath-hold techniques may be applied for the entire DKI acquisition without causing unfeasible scan times. One common pathological condition for which body DKI may be of immediate clinical value is kidney fibrosis, which causes progressive changes in organ microstructure. With its sensitivity to microstructure, DKI is an obvious candidate for a non-invasive evaluation method. We present preclinical evidence indicating that the rapidly obtainable tensor-derived mean kurtosis ( W̅) distinguishes moderately fibrotic kidneys from healthy controls. The presence and degree of fibrosis are confirmed by histology, which also indicates fibrosis as the main driver behind the DKI differences observed between groups. We therefore conclude that fast kurtosis is a likely candidate for an MRI-based method for the detection and monitoring of renal fibrosis. We provide protocol recommendations for fast renal DKI in humans based on a b-value optimisation performed using data acquired at 3 T in normal human kidney.

Stereological estimation of particle shape and orientation from volume tensors.

In the present paper, we describe new robust methods of estimating cell shape and orientation in 3D from sections. The descriptors of 3D cell shape and orientation are based on volume tensors which are used to construct an ellipsoid, the Miles ellipsoid, approximating the average cell shape and orientation in 3D. The estimators of volume tensors are based on observations in several optical planes through sampled cells. This type of geometric sampling design is known as the optical rotator. The statistical behaviour of the estimator of the Miles ellipsoid is studied under a flexible model for 3D cell shape and orientation. In a simulation study, the lengths of the axes of the Miles ellipsoid can be estimated with coefficients of variation of about 2% if 100 cells are sampled. Finally, we illustrate the use of the developed methods in an example, involving neurons in the medial prefrontal cortex of rat.

Detection and spatial characterization of minicolumnarity in the human cerebral cortex.

BACKGROUND: Spatial characterization of vertical organization of neurons in human cerebral cortex, cortical columnarity or minicolumns, and its possible association with various psychiatric and neurological diseases has been investigated for many years. NEW METHOD: In this study, we obtained 3D coordinates of disector sampled cells from layer III of Brodmann area 4 of the human cerebral cortex using light microscopy and 140-µm-thick glycolmethacrylate sections. A new analytical tool called cylindrical K-function was applied for spatial point pattern analysis of 3D datasets to see whether there is a spatially organized columnar structure. In order to demonstrate the behaviour of the cylindrical K-function, the result from brain tissues was compared with two models: A homogeneous Poisson process exhibiting complete spatial randomness, and a Poisson line cluster point process. The latter is a point process model in 3D space, which exhibits spatial structure of points similar to minicolumns. RESULTS: The data show in three out of four samples nonrandom patterns in the 3D neuronal positions with the direction of minicolumns perpendicular to the pial surface of the brain - without a priori assuming the existence of minicolumns. COMPARISON WITH EXISTING METHODS: Studies on columnarity are difficult and have mainly been based on two-dimensional images analysis of thin sections of the cerebral cortex with the a priori assumption that minicolumns existed. CONCLUSIONS: A clear difference from complete spatial randomness in the data could be detected with the new tool, the cylindrical K-function, although classical functional summary statistics are less useful in this connection.

Stress and corticosterone increase the readily releasable pool of glutamate vesicles in synaptic terminals of prefrontal and frontal cortex.

Stress and glucocorticoids alter glutamatergic transmission, and the outcome of stress may range from plasticity enhancing effects to noxious, maladaptive changes. We have previously demonstrated that acute stress rapidly increases glutamate release in prefrontal and frontal cortex via glucocorticoid receptor and accumulation of presynaptic SNARE complex. Here we compared the ex vivo effects of acute stress on glutamate release with those of in vitro application of corticosterone, to analyze whether acute effect of stress on glutamatergic transmission is mediated by local synaptic action of corticosterone. We found that acute stress increases both the readily releasable pool (RRP) of vesicles and depolarization-evoked glutamate release, while application in vitro of corticosterone rapidly increases the RRP, an effect dependent on synaptic receptors for the hormone, but does not induce glutamate release for up to 20 min. These findings indicate that corticosterone mediates the enhancement of glutamate release induced by acute stress, and the rapid non-genomic action of the hormone is necessary but not sufficient for this effect.

Growth hormone mitigates loss of periosteal bone formation and muscle mass in disuse osteopenic rats.

Growth hormone (GH) is a potent anabolic agent capable of increasing both bone and muscle mass. The aim was to investigate whether GH could counteract disuse-induced loss of bone and muscle mass in a rat model. Paralysis was induced by injecting 4 IU Botox (BTX) into the muscles of the right hind limb. Sixty female Wistar rats, 14 weeks old, were divided into the following groups: baseline, controls, BTX, BTX+GH, and GH. GH was given at a dosage of 5 mg/kg/d for 4 weeks. Compared with controls, BTX resulted in lower periosteal bone formation rate (BFR/BS,-79%, P<0.001), bone mineral density (aBMD, -13%, P<0.001), trabecular bone volume (BV/TV, -26%, P<0.05), and mid-femoral bone strength (-12%, P<0.05). In addition, BTX reduced rectus femoris muscle mass (-69%, P<0.001) and muscle cell cross sectional area (CSA) (-73%, P<0.001) compared with controls. GH counteracted disuse-induced losses of periosteal BFR/BS (2-fold increase vs. BTX, P<0.001), whereas no effect on aBMD, trabecular BV/TV, or bone strength was found. In addition, GH partly prevented loss of muscle mass (+29% vs. BTX, P<0.001), and tended to prevent loss of muscle CSA (+11%, P=0.064). In conclusion, GH mitigates disuse-induced loss of periosteal BFR/BS at the mid-femur and rectus femoris muscle mass.

Improving efficiency in stereology: a study applying the proportionator and the autodisector on virtual slides.

Cell counting in stereology is time-consuming. The proportionator is a new stereological sampling method combining automatic image analysis and non-uniform sampling. The autodisector on virtual slides combines automatic generation of disector pairs with the use of digital images. The aim of the study was to investigate the time efficiency of the proportionator and the autodisector on virtual slides compared with traditional methods in a practical application, namely the estimation of osteoclast numbers in paws from mice with experimental arthritis and control mice. Tissue slides were scanned in a digital slide scanner and the autodisector was applied on the obtained virtual tissue slides. Every slide was partitioned into fields of view, and cells were counted in all of them. Based on the original exhaustive data set comprising 100% of fields of view and covering the total section area, a proportionator sampling and a systematic, uniform random sampling were simulated. We found that the proportionator was 50% to 90% more time efficient than systematic, uniform random sampling. The time efficiency of the autodisector on virtual slides was 60% to 100% better than the disector on tissue slides. We conclude that both the proportionator and the autodisector on virtual slides may improve efficiency of cell counting in stereology.

The spatial rotator.

This paper presents a new local volume estimator, the spatial rotator, which is based on measurements on a virtual 3D probe, using computer assisted microscopy. The basic design of the probe builds upon the rotator principle which requires only a few manual intersection markings, thus making the spatial rotator fast to use. Since a 3D probe is involved, it is expected that the spatial rotator will be more efficient than the the nucleator and the planar rotator, which are based on measurements in a single plane. An extensive simulation study shows that the spatial rotator may be more efficient than the traditional local volume estimators. Furthermore, the spatial rotator can be seen as a further development of the Cavalieri estimator, which does not require randomization of sectioning or viewing direction. The tissue may thus be sectioned in any arbitrary direction, making it easy to identify the specific tissue region under study. In order to use the spatial rotator in practice, however, it is necessary to be able to identify intersection points between cell boundaries and test rays in a series of parallel focal planes, also at the peripheral parts of the cell boundaries. In cases where over- and underprojection phenomena are not negligible, they should therefore be corrected for if the spatial rotator is to be applied. If such a correction is not possible, it is needed to avoid these phenomena by using microscopy with increased resolution in the focal plane.

Application of design-based stereology for estimation of absolute volume and surface area of the articular and calcified cartilage compartments of undecalcified human femoral heads.

Design-based stereological methods using systematic uniform random sampling, the Cavalieri estimator and vertical sections are used to investigate undecalcified human femoral heads. Ten entire human femoral heads, obtained from normal women and normal men, were systematically sampled and thin undecalcified vertical sections were obtained. Absolute volumes and surface areas of the entire femoral head, the articular cartilage and the calcified cartilage compartments were estimated. In addition, the average thickness of the articular cartilage and the calcified cartilage were calculated. The stereological procedures applied to the human femoral heads resulted in average coefficient of errors, which were 0.03-0.06 for the volume estimates and 0.03-0.04 for the surface area estimates. We conclude that design-based stereology using the Cavalieri estimator and vertical sections can successfully be used in large undecalcified tissue specimens, like the human femoral head, to estimate the absolute volume and surface area of macroscopic as well as of microscopic tissue compartments. The application of well-known design-based stereological methods carries potential advantage for investigating the pathology in inflammatory and degenerative joint diseases.

The semi-automatic nucleator.

The nucleator is a well-established manual stereological method of estimating mean cell volume from observations on random cell transects through reference points of the cells. In this paper, we present an automated version of the nucleator that uses automatic segmentation of the boundaries of the cell transects. An expert supervises the process. If the segmentation is judged to be satisfactory, an estimate of the cell volume is calculated automatically on the basis of the whole cell transect. In the remaining cases, the expert intervenes and uses the classical nucleator. The resulting estimator is called the semi-automatic nucleator. In this paper, we study the statistical properties of the semi-automatic nucleator. Formulae for the bias and mean square error are derived. The semi-automatic nucleator may have a small bias but will still in most cases be more efficient than the classical nucleator. Procedures for estimating bias and mean square error from a pilot study are provided. The application of the semi-automatic nucleator is illustrated in a study of somatostatin positive inhibitory interneurons which were genetically labelled with green fluorescent protein (GFP). The cells were sampled with an optical disector. The centre of mass in a central cell transect was used as reference point. It is found in this study that the number of cells needed for obtaining, for instance, a 5% precision of the estimate of mean cell volume is 150 and 189 for the semi-automatic and the classical nucleator, respectively. Taking into account that the time spent analysing one cell is shorter for the semi-automatic nucleator than for the classical nucleator, the semi-automatic nucleator is superior to the classical nucleator.

Histological changes in chronic autoimmune SKG-arthritis evaluated by quantitative three-dimensional stereological estimators.

OBJECTIVES: To investigate the quantitative arthritic and bone erosive changes, including the number of osteoclasts and osteoclast precursors in the new SKG-model of inflammatory polyarthritis using three-dimensional (3D) stereological methods. METHODS: Arthritis was induced in female SKG-mice with Zymosan A. Quantitative histology was made in four control mice and four mice with arthritis euthanised after 6 and 12 weeks. The right hind paw was embedded undecalcified in methylmethacrylate and cut exhaustively generating vertical uniform random sections. A computer controlled microscope and stereological software was used for histological quantification. Total volumes were estimated according to the Cavalieri principle, total surfaces were estimated using the vertical sections design, and the number of osteoclasts was counted in a physical fractionator. RESULTS: The arthritis score increased during the 12-week period and was paralleled by an increase in the volume of inflammatory tissue (r=0.96, p<0.001). The number of osteoclasts on bone (r=0.77, p<0.05) and osteoclast-covered bone surface (r=0.62, p<0.05) increased resulting in a decrease in the volume of bone (r=-0.65, p<0.05). However, the number of osteoclast precursors declined between week 6 and 12 (p<0.05). Furthermore, the total cartilage surface (r=-0.74, p<0.05) and cartilage volume (r=-0.74, p<0.05) decreased during the 12 weeks of arthritis. CONCLUSIONS: In this study we demonstrated changes in 3D stereological parameters of inflammatory tissue, bone erosion, osteoclasts, and cartilage in mouse paws during the course of arthritis in the SKG mouse. This is the first time 3D quantitative histology has been applied in a mouse model of rheumatoid arthritis.

Stereological estimation of the total number of ventilatory units in mice lungs.

We present an unbiased estimator of the total number of alveolar structures distal to the transition from a bronchiole to an alveolar duct system ('ventilatory units', VUs). In species without respiratory bronchioles, including mice, the number of VUs is equivalent to the number of acini. The acinus is a functional unit of gas exchange, defined as a parenchymal unit distal to a terminal bronchiole in which all airways contain alveoli and thus participate in gas exchange. The estimator combines two different estimators of the number of VUs: (1) an estimator derived from the Euler number of all the openings of the bronchial tree and (2) an estimator derived from direct counts of topological changes occurring at bronchiole-alveolar duct junctions. Combining the two estimators eliminates the requirement to be able to identify even vanishingly small pieces of bronchial tissue in physical disectors. We implemented the fractionator estimator in five adult mice lungs using physical fractionators with varying but known sampling fractions (Horvitz-Thompson estimator). We obtained total values of about 4200 VUs (CV = 0.05) in 21-day-old and 4480 (CV = 0.06) in 69-day-old animals. Being fractionator estimates, these total numbers are independent of shrinkage. The densities of VUs per unit volume of tissue (values corrected for tissue shrinkage) were similar in left and right lungs.

Estimation of variance components of local stereological volume estimators: a pilot study.

Local stereological techniques can be used for particle volume estimation based on information collected on a section plane through a reference point of the particle. We present methods for variability estimation of the local stereological volume estimators. This variability arises during the stereological estimation procedure and in the particle population. Both of these components can be estimated separately from planar sections. Our aim is to give a preliminary analysis of the possibility to include the particle structure interaction into the estimation procedure. For this reason, not only the section profiles, but also their locations, have to be recorded. The methods are applied for the sectional data obtained from neurons in the hippocampal brain region subiculum of four 3-month-old male Wistar rats. The proposed procedure enables one to obtain information about particle volume distribution.

Application of stereological principles for quantification of bacteria in intact dental biofilms.

INTRODUCTION: Quantative confocal laser scanning microscopy (CLSM) in combination with fluorescent in situ hybridization (FISH) may help to increase our knowledge about biofilm formation. The purpose of this study was to develop and evaluate a stereological method for quantification of bacteria in intact biofilm. The method was applied in a quantitative study of the proportion of streptococci relative to other bacteria in initial in-situ-grown dental biofilms as a function of time. METHODS: Biofilms were collected on standardized glass slabs mounted in intra-oral appliances and worn by 10 individuals for 6, 12, 24, and 48 h. Biofilms were analysed using CLSM. Quantification of bacteria labelled with 16S ribosomal RNA oligonucleotide probes was performed with stereological tools: the unbiased counting frame and the two-dimensional fractionator. RESULTS: Results showed a notable increase in the total number of bacteria and streptococci over time, with a considerable inter-individual variation at each time-point. After 48 h there was a 12.5-fold difference between individuals in the total number of bacteria and a 12.6-fold difference in the number of streptococci. The number of streptococci exceeded that of other bacteria and over the examination period there was a relatively constant relationship between the number of streptococci and other bacteria (streptococci vs. non-streptococci: median 15.2; minimum 1.0; maximum 89.3). CONCLUSION: The study demonstrates that the combined use of FISH and stereology is a relevant and reliable tool for obtaining unbiased information about the numerical contributions of specific bacterial populations during early biofilm formation.

Trigeminal nociceptive function and oral somatosensory functional and structural assessment in patients with diabetic peripheral neuropathy.

This case-control study primarily compared the trigeminal nociceptive function, the intraoral somatosensory profile and possible structural nerve changes between diabetic peripheral neuropathy (DPN, n = 12) patients and healthy participants (n = 12). The nociceptive blink reflex (nBR) was recorded applying an electrical stimulation over the entry zone of the right supraorbital (V1R), infraorbital (V2R) and mental (V3R) and left infraorbital (V2L) nerves. The outcomes were: individual electrical sensory (I0) and pain thresholds (IP); root mean square (RMS), area-under-the-curve (AUC) and onset latencies of R2 component of the nBR. Furthermore, a standardized full battery of quantitative sensory testing (QST) and intraepidermal nerve fibre density (IENFD) or  nerve fibre length density (NFLD) assessment were performed, respectively, on the distal leg and oral mucosa. As expected, all patients had altered somatosensory sensitivity and lower IENFD in the lower limb. DPN patients presented higher I0, IP, RMS and AUC values (p < 0.050), lower warm detection thresholds (WDT) (p = 0.004), higher occurrence of paradoxical heat sensation (PHS) (p = 0.040), and a lower intraoral NFLD (p = 0.048) than the healthy participants. In addition, the presence of any abnormal intraoral somatosensory finding was more frequent in the DPN patients when compared to the reference group (p = 0.013). Early signs of trigeminal nociceptive facilitation, intraoral somatosensory abnormalities and loss of intraoral neuronal tissue can be detected in DPN patients.