String Vessel Formation is Increased in the Brain of Parkinson Disease

Abstract

Background:

String vessels are collapsed basement membrane without endothelium and have no function in circulation. String vessel formation contributes to vascular degeneration in Alzheimer disease. By comparing to age-matched control cases we have recently reported endothelial degeneration in brain capillaries of human Parkinson disease (PD).

Objective:

Current study evaluated changes of basement membrane of capillaries, string vessel formation and their association with astrocytes, blood-brain-barrier integrity and neuronal degeneration in PD.

Methods:

Brain tissue from human cases of PD and age-matched controls was used. Immunohistochemical staining for collagen IV, GFAP, NeuN, tyrosine hydroxylase, fibrinogen and Factor VIII was evaluated by image analysis in the substantia nigra, caudate nucleus and middle frontal gyrus.

Results:

While the basement-membrane-associated vessel density was similar between the two groups, the density of string vessels was significantly increased in the PD cases, particularly in the substantia nigra. Neuronal degeneration was found in all brain regions. Astrocytes and fibrinogen were increased in the caudate nuclei of PD cases compared with control cases.

Conclusions:

Endothelial degeneration and preservation of basement membrane result in an increase of string vessel formation in PD. The data may suggest a possible role for cerebral hypoperfusion in the neuronal degeneration characteristic of PD, which needs further investigation. Elevated astrocytosis in the caudate nucleus of PD cases could be associated with disruption of the blood-brain barrier in this brain region.

INTRODUCTION

Parkinson’s disease (PD) is a neurological condition characterized by progressive degeneration of dopamine neurons [1]. No curative therapy exists [2]. Neuronal degeneration can spread into other brain regions as the condition advances [3]. Vascular degeneration is a recognized contributing factor to Alzheimer disease [4, 5] but there is limited information on vascular changes in PD and their association with disease progression. We have recently reported, for the first time, that there is endothelial degeneration in several brain regions of humans with PD [6]. Using an antibody against Factor VIII as a marker for endothelial cells, we demonstrated that there are fewer capillaries in the brains of PD cases, and the capillaries are shorter and larger in diameter with fewer connecting branches compared to age-matched controls [6]. This PD-associated vascular degeneration was identified in brain regions with dopamine neuron degeneration, for example the substantia nigra (SN) and brain stem nuclei, but not in the caudate nucleus (CN) [6], a region that showed severe age-associated capillary loss in control tissue. Clear vascular degeneration was also found in brain regions where neuronal degeneration was not specific to dopamine neurons, for example the middle frontal gyrus (MFG) [6]. These particular changes in various brain regions may be due to differences between PD-specific pathology and the combined effects of brain aging and the secondary pathology of PD.

Apart from the endothelium, brain capillaries also consist of several outer layers, including basement membrane (BM), astrocytic endfeet and pericytes [7]. These components are critical for capillary structure and for the function of the capillaries and the blood brain barrier (BBB) [7]. The BM provides the extracellular matrix scaffold for the capillary endothelium and produces growth factors for maintaining vascular remodelling [8] and neuronal migration and development [9].

String vessels are formed by collapsed BM and have been described as thin connective tissue strands, remnants of capillaries, with an absence of endothelial cells [10]. String vessels have no function in blood flow. The formation of string vessels has been suggested to be the result of incomplete endothelial turnover during the process of vascular remodelling. String vessels are present in the normal brain throughout life, but do not increase with normal aging [10]. The increase in string vessels during early stages of brain development may possibly be due to rapid turnover of endothelial cells [10]. Increased numbers of string vessels have been also found under certain pathological conditions, for example Alzheimer disease [11] but there is no information on string vessels in PD.

Apart from forming the outer layer of capillaries, astrocytes play critical roles in angiogenesis and cell-cell communication between neurons and endothelial cells [12]. Together with BM, astrocytes provide trophic factors to maintain BBB integrity and functions, as well as vascular remodelling [13]. Both insufficient vascular remodelling and compromised BBB function contribute to neuronal dysfunction and degeneration [7].

To further study capillary degeneration and its relationship to the progression of PD, we evaluated changes in the BM and astrocytes, and their association with endothelial and neuronal degeneration and the integrity of the BBB, in human brains by comparison of PD cases and aged-matched controls.

METHODS

The study has been approved by University of Auckland Human Participants Ethics Committee(Reference number 2008/279).

Tissue preparation

Tissue preparation has been described previously [14]. Briefly, we used tissue samples from the Neurological Foundation New Zealand Human Brain Bank. Brains had been perfused through the cerebral arteries with phosphate buffered saline (PBS) containing 1% sodium nitrite for 15 minutes followed by 15% formalin solution in 0.1M PBS for 30–45 minutes and then placed in the same formalin fixative solution overnight. The brains were then dissected into blocks which were post-fixed for a further one to two days before being cryoprotected prior to freezing. Cryoprotection was completed by transfer of the blocks sequentially into a 20% and then 30% sucrose solution in 0.1M PBS with 0.1% sodium azide. At this point, the blocks were stored at –80°C until required.

Case selection

Brain tissue from 9 PD cases (Table 1) and 6 age-matched control cases (Table 2) was obtained. Selection of PD cases was based on the following selection criteria: a post-mortem delay less than 48 hours and a clinical diagnosis of idiopathic PD. For the pathological diagnosis a neuropathologist examined several key diagnostic regions of the brains all of the PD and control cases. The anatomical areas sampled included the substantia nigra, pons at the level of locus coeruleus, middle frontal gyrus, middle temporal gyrus, hippocampus with the entorhinal cortex, caudate nucleus/putamen, cingulate gyrus, inferior parietal lobule, occipital cortex and cerebellum. Tissue blocks were processed for paraffin embedding, 4 micron thick tissue sections were cut, which were subsequently stained with haematoxylin and eosin, and immunohistochemistry was performed with antibodies to tau (DAKO A0024), beta-amyloid (DAKO 6F/3D), and alpha-synuclein (Leica clone KM51). The extent of Lewy Body disease was staged according to the McKeith type [15] using the protocol suggested by the BrainNet Europe consortium [16]. All cases were assessed for Alzheimer disease neuropathologic change according to the current NIA/AA guidelines [17]. The phase of beta amyloid aggregation (Thal phase) was derived using the method outlined by the BrainNet Europe Consortium [18]. The Braak stage was assessed using tau immunohistochemistry according to Alafuzoff et al. [19]. The neuritic plaque density was estimated with a modified Bielschowsky stain. The age-matched controls had no clinical history of neurological or dementing illness and any Alzheimer disease neuropathologic change had a maximum Braak stage of II [17, 20]. In two control cases alpha-synuclein staining revealed very low level Lewy body disease in the brain stem and amygdala (see Table 1). Given the lack of clinical disease these cases were included in the final analysis.

Immunohistochemical staining

A detailed protocol of the methods used has been published recently [6, 21]. Briefly, sequential coronal sections (50 μm) of MFG, the CN of the striatum and SN were collected and used. Three sections (12 sections and 600 μm apart) were chosen from each region per case. Immunohistochemistry of Von Willebrand-associated Factor VIII (Factor VIII) for endothelial cells, collagen IV for basement membrane, GFAP for astrocyte, NeuN for neurons and tyrosine hydroxylase (TH) for dopamine neurons were conducted The free floating methodology was employed for immunohistochemical staining; this has been described previously [6, 21]. All washes, unless specifically noted, were carried out three times for 10 minutes each in PBST (PBS with 0.2% Triton X-100 (v/v)) with gentle shaking. All antibodies were diluted with immunobuffer (1% goat serum (v/v) and 0.4% merthiolate (w/v) in PBST).

The selected sections were incubated with PBST overnight at 4°C before they were pre-treated with a solution of 50% methanol (v/v) and 1% hydrogen peroxide (v/v) for 20 minutes. The sections were then washed before being incubated with primary antibodies (Table 3) for 48 hours at 4°C. The tissue was then washed and incubated with biotinylated secondary antibodies (Table 3) for 24 hours at room temperature, followed by washes and incubation with ExtrAvidin-HRP (Table 3) for four hours at room temperature. The tissue was then washed and incubated in a solution of 0.05% DAB (w/v) with 0.01% hydrogen peroxide (v/v) in 0.1 M phosphate buffer for 15 minutes to give a brown reaction product. Sections were then mounted onto glass slides, air-dried, dehydrated through a graded alcohol series and cover-slipped. Negative control staining was generated by omitting primary antibody.

For fluorescent immunohistochemistry, sections were incubated with PBST overnight followed by incubation in mouse anti-collagen IV and rabbit anti-Factor VIII antibodies (Table 4) for 48 hours at 4°C. The sections were washed before they were incubated with secondary goat anti-mouse IgG Alexa 647 and goat anti-rabbit IgG Alexa 594 (Table 4) overnight at 4°C. Following three 10-minute PBST washes, a blocking step was applied by incubating the sections with ChromPure Mouse IgG (1:10, Jackson Immunoresearch Laboratories, West Grove, PA, USA) diluted with PBST for 4 hours at room temperature. The sections were then washed in PBST and incubated with mouse anti-GFAP antibody Alexa 488 conjugate (Table 4) for 48 hours at 4°C. Following this, the sections were subjected to nuclear staining by 20-minute incubation in Hoechst stain (1:10,000, Molecular Probes, Eugene, OR, USA) and auto-fluorescence reduction by 5-minute incubation in 0.1% Sudan Black B in 70% ethanol. The sections were then briefly washed by two 2-minute PBST washes, mounted onto glass slides with PBS, cover-slipped with Prolong Gold antifade reagent (Molecular Probes, Eugene, OR, USA), and sealed with nail polish. The slides were stored overnight at 4°C in the dark before imaging.

Image processing and analysis

Staining of collagen IV, NeuN, TH and fibrinogen

The methods used for image analysis have been reported previously [6]. Images were captured with a 20×objective on a Nikon E800 microscope equipped with a digital camera (mbf Bioscience) and a motorised stage to enable randomised sampling of images from the regions of interest. Twenty images were obtained from each section of SN, CN and MFG by randomised sampling. Each sample area was approximately 0.26 mm². MetaMorph^TM software (Molecular Devices) was used to analyse the number and length of the BM of blood vessels, and the number and intensity of NeuN positive and TH positive neurons, in the acquired images. Macros were developed in MetaMorph^TM to enable automated high throughput processing of large numbers of images. The macros first converted the images into 16-bit format using the “Arithmetic” tool and inverted them to grayscale pixel values using the “Morphological Invert” tool so that positive staining had higher pixel intensity values than the background. For the BM analysis, the images were processed with the “Angiogenesis Tube Formation” algorithm and parameters including the connected sets and total tubule length were selected for summary logs to record the number of connected blood vessels and the total blood vessel length, respectively. The number and length of string capillaries were not processed by a macro because string capillaries cannot be consistently recognised in each image by applying one setting to all images. Therefore, instead of automated image processing, the “Multi-line” tool was used to manually trace the string capillaries in order to obtain information regarding the number and length of the capillaries. All the images were blinded for manual quantification. For the NeuN positive neuron analysis, the images were processed with the “Count Nuclei” algorithm and parameters including the total number and integrated intensity were selected for summary logs. For the TH positive neuron analysis, the “Morphological Erode” tool was used prior to image processing to eliminate the fine processes from neurons so that only cell bodies were analysed. The images were then processed by the same approach as for NeuN positive neuron analysis and the same parameters were recorded.

Fibrinogen

Images of the whole tissue section were captured at 0.8×magnification on a Leica MZ6 modular stereomicroscope equipped with a Nikon Digital Sight DS-SM digital camera. The intensity of the fibrinogen staining in the regions of interest in these images was measured using ImageJ software (V1.46). The region of interest in an image was marked by the “Freehand Selections” tool. The image was then converted into 8-bit format and the grayscale pixel values in the region of interest were inverted so that the positive staining had higher pixel intensity values than the background. The intensity information of the region of interest was obtained by using the “Measure” tool.

Staining of GFAP

The methods used for analysing astrocytes have been reported previously [6]. Briefly, the images were captured using a 10×objective on the Leica DMRB microscope equipped with a digital camera (Nikon Digital Sight DS-SM). Four images were acquired from each section of each region of interest. Each sample area was approximately 8.325 mm². ImageJ software (V1.46) was used to measure the number and area of the GFAP positive astrocytes in these images. The images were converted into 8-bit format and the “Subtract Background” tool was used to reduce background and lighten the images. The GFAP positive astrocytes in the image were selected using the “MaxEntropy” auto-threshold function in the “Threshold” tool before a binary image was created. The binary images were then processed using the “Analyse Particle” tool, which provides information regarding the number and coverage of the GFAP positive astrocytes.

Statistical analysis

The difference between the age-matched control and PD cases was analyzed in the various brain regions using a two-way ANOVA with brain regions treated as dependent factors. The Bonferroni post-test was used for specific differences between the individual brain regions. The Spearman test was used for analysis of correlation between the neurons/astrocytes and string vessels.

RESULTS

Changes in collagen IV positive capillaries

The BM was visualised using collagen IV staining. Two-way ANOVA showed that both total length and density of collagen IV positive capillaries were similar between the PD cases and age-matched controls in all three brain regions examined (Fig. 1A & B). Morphologically, some parts of the BM were narrow and had lost their tubular morphology (Fig. 2). Triple labelling of collagen IV (for BM, white), Factor VIII (for endothelial cells, red) and GFAP (for astrocytes, green) showed that the narrow vessels were collapsed BM with no endothelial cells (Fig. 2 A-C), which have been described as string capillaries by others [10]. The endothelial cells of the capillaries connected to the string vessels showed a clear laddering morphology (Fig. 2C), characterised as endothelial clusters in our previous report [6]. The photomicrographs in Fig. 3 show the distributions and morphologies (inserts) of the string capillaries (arrows) in both age-matched control and PD cases in the SN (Fig. 3A, B), CN (Fig. 3C, D) and MFG (Fig. 3 E, F). Visual observation indicated that there were obviously more string capillaries in the PD cases compared to the age-matched control cases (Fig. 3). The density and total length of string capillaries were therefore measured using image analysis software.

Two-way ANOVA showed a significant difference in the density of string capillaries between the two groups (F[1,37, 1,37] = 22.79, p <  0.0001, Fig. 4A). There were no differences among the brain regions and no interactions between the groups and the brain regions. Multiple comparison showed that the string capillary density was significantly increased in the SN (p <  0.01) and MFG (p <  0.05). The increase in CN was not statistically significant in the PD cases compared to the age matched controls (p >  0.05).

The total length of string capillaries was also significantly different between the two groups (F[1,37, 1,37] = 22.28, p <  0.0001, Fig. 4B) and among the brain regions (F[2.37] = 3.60, p = 0.03) with no interactions between the groups and brain regions. Multiple analysis showed that the total length of string capillaries was significantly increased in the SN of the PD cases compared to the control cases (p <  0.01). There was a strong trend toward an increase in string capillaries in the MFG and CN, but the difference was not statistically significant.

Changes in GFAP positive astrocytes

Photomicrographs (Fig. 5) show the distributions and morphologies (inserts) of astrocytes in the SN, CN and MFG of both age-matched controls and PD cases. In the SN, the astrocytes were distributed amongst the darkly stained large neurons, presumably the substantia nigra pars compacta dopamine neurons. Morphologically, the processes and cytoplasms of GFAP staining in the SN were similar between the two groups. There was no obvious sign of hypertrophied morphology (Fig. 5A, B). However, compared to the age-matched controls, the astrocytes in the CN of the PD cases showed hyperactive morphology with enlarged and densely stained cytoplasm (Fig. 5C, D, inserts). The reactive astrocytes were densely distributed in the CN of the PD cases (Fig. 5C, D). In the MFG, the distribution and morphology of GFAP positive astrocytes were similar between the age-matched controls and PD cases (Fig. 5E, F).

Two way ANOVA showed that the density of astrocytes was significantly different between the two groups (F[1,30, 1,30] = 8.56, p = 0.0065, Fig. 6A) and there was a significant interaction between the groups and brain regions (F[2,30, 2,30] = 8.37, p = 0.0013). Multiple comparison showed that the density of astrocytes was significantly increased in the CN of the PD cases compared to the age-matched controls (p <  0.001, Fig. 6A). The density of astrocytes in the SN and MFG was not significantly different between the two groups.

We also found a significant difference in the percentage of area staining among the groups (F[2,35, 2,35] = 16.48, p <  0.0001) and there was a significant interaction between the groups and brain regions (F[2,35, 2,35] = 4.41, P = 0.019). Multiple comparisons showed a significant increase in the area with GFAP staining in the CN of PD cases compared to the age-matched controls (p <  0.05). The areas of staining were not significantly different between the SN and MFG.

Changes in neurons

Figure 7 shows the distribution and morphology of TH (A & B) and NeuN (C-F) positive neurons in the SN, CN and MFG of both age-matched controls and PD cases. Compared to the controls, there were fewer but more densely stained TH positive neurons in the PD cases. Both the density of NeuN positive neurons and the staining intenstiy of NeuN were obviously reduced in both the CN and MFG of the PD cases compared to the controls.

The density (Fig. 8A) and the intensity (Fig. 8B) of TH positive dopamine neurons in the SN were analysed by the two-tailed t-test. Compared to the age-matched controls, the density of TH positive dopamine neurons were significantly reduced in the PD cases (P <  0.05), as expected. In contrast, the average intensity of TH staining was increased in the PD cases compared to the controls (p <  0.05).

The density (Fig. 9A) and the intensity (Fig. 9B) of NeuN positive neurons were analysed in the CN and MFG by two-way ANOVA. Compared to the age-matched controls, the density of neurons was significantly reduced in the PD cases (F[1,24, 1,24] = 12.45, p = 0.0017, Fig. 9A). There were no differences between brain regions and no interactions between brain regions and groups.

The average NeuN staining intensity in neurons was also reduced in PD cases compared to controls (F[1,24, 1,24] = 15.14, p = 0.0007, Fig. 9B). The effects were not dependent on brain region. Multiple comparisons showed a significant decrease in the intenstiy of NeuN staining in the CN (p <  0.05) and MFG (p <  0.05) of PD cases.

Changes in fibrinogen intensity

Fibrinogen was densely stained in the substantia nigra of both the PD cases and the age-matched controls (Fig. 10, top panel), particularly around the blood vessels. The pattern and the distribution of the staining were similar between the PD cases and the age-matched controls. However the area of staining was larger in the CN of the PD cases compared to the controls (Fig. 10, middle panel). The pattern and distribution of fibrinogen staining were similar between the two groups in the MFG (Fig. 10, bottom panel). The average intensity of fibrinogen was compared between the two groups. Two-way ANOVA suggested a difference between the groups (F[1,32, 1,32] = 9.90, p = 0.003, Fig. 11A) and among the brain regions (F[2,32, 2,32] = 18.67, p <  0.0001), with no interactions between groups and brain regions. Multiple analysis showed a specific increase in the intensity of fibrinogen only in the CN (p <  0.05), but not in the SN or MFG. The maximum intensity was also increased in the PD cases (F[1,32, 1,32] = 4.48, p = 0.042, Fig. 11B) with a specific up-regulation in the CN region (p <  0.01).

DISCUSSION

Our results have demonstrated that increased string vessel formation in PD is linked to the degeneration of endothelial cells and preservation of BM, and is found in brain regions associated with neuronal degeneration. Thus, the data suggest a potential role for brain hypoperfusion in the progression of PD. The elevated astrocytosis that suggests an association with dysfunction of the BBB in the CN could be an angiogenic response to endothelial degeneration rather than a pathological change specific to PD.

Using a marker for endothelial cells, we have previously reported endothelial degeneration in PD by showing loss of capillary density and capillary networks [6]. Given that the structure and functions of capillaries are complex, we have now further characterized this capillary degeneration by investigating changes in BM, astrocytes and BBB integrity. We evaluated the changes in BM in the same human cases used for evaluating endothelial cell degeneration. Unexpectedly, we found no difference in either the density or the total length of collagen IV labelled capillaries between age-matched controls and PD cases, the same parameters we have previously analysed to identify endothelial degeneration [6]. However, we observed profound morphological changes in the BM, characterized as collapsed capillaries or string vessels [10], in which endothelial cells were largely absent from the collapsed collagen IV labelled vessels. String capillaries exist in normal brain as part of the process of endothelial cell turnover during vascular remodelling, and can increase during brain development, possibly as a result of rapid and active vascular remodelling [10]. In addition, the increase in string vessels that is seen under certain pathological conditions with the absence of endothelial cells within capillaries could be the result of endothelial cell degeneration and/or insufficient endothelial cell proliferation and migration during vascular remodelling [10]. Capillaries with degenerative endothelium indicated by the laddering morphology of endothelial staining [6] were often found next to the string vessels. This also suggests that string vessel formation may be a consequence of endothelial degeneration.

Even though the function of string vessels is not yet fully characterised, they do not carry blood flow and have no function as capillaries [10]. An increase in string vessels has been reported in the white matter [5] and other brain regions [3] of Alzheimer disease cases; this pathology appears to be characterized by a profound loss of BM in brain regions with increased string vessels [11]. In contrast, capillary density has recently been reported to be increased in human cases of Huntington’s disease when collagen IV was used for labelling the BM [22]. Thus, increased stringvessel formation without loss of BM may be a pathology specific for vascular degeneration in PD.

There has been a rapid increase in research on changes in capillaries in brains affected by neurological diseases. It appears that different markers used for labelling different structures of the capillary can lead to dissimilar conclusions. For example, there were no PD-associated changes in capillary density when the BM was labelled by collagen IV, whereas a reduction in vascular density was found when endothelial cells were labelled by Factor VIII [6]. For an objective view of vascular changes, it is essential to use multiple markers specific for the various components of capillaries rather than a single marker.

It has been suggested that string vessels represent an individual record of the history of the microcirculation [10], in which capillaries are no longer functional because of profound endothelial cell degeneration, and/or are not fully developed because of insufficient endothelial proliferation and migration during vascular remodelling. In any case, increased string vessel formation may indicate a compromised blood supply that may contribute to neuronal dysfunction and degeneration. As extracellular matrix, BM plays a critical role in vascular remodelling, for example promoting endothelial cell proliferation and migration and capillary sprouting [8]. However, structurally maintained BM does not necessarily imply that vascular remodelling is sustained in brain regions showing increased string vessel formation; this needs to be investigated.

As a component of capillaries, astrocytes also play a critical role in vascular remodelling [13, 23] and neuronal protection [24, 25]. Using GFAP as a marker for reactive astrocytes, we found a massive elevation of astrocytosis in the CN, where the astrocytes showed hyperactive morphology. Our study suggests that PD-associated endothelial degeneration in the CN is the result of age, rather than a change specific to PD, as we do not see a further loss of endothelial-associated capillaries when comparing PD cases and age-matched controls [6]. Given that the endothelial changes were evaluated in the same brain samples as those that showed the increase in reactive astrocytes in the CN, we suggest that the elevated astrocytosis may be a protective and/or angiogenic response that prevented further loss of endothelial cells in PD cases [6]. Angiogenesis is a double edged sword as newly formed vessels can lead to BBB leakage, which occurs in neurodegenerative conditions such as Alzheimer’s disease [26]. However it is not clear whether or not BBB dysfunction contributes to neuronal degeneration in the CN.

The leakage of large molecules, for example plasma fibrinogen, to brain parenchyma is an effective marker for dysfunction of the BBB, which has been well described in Alzheimer’s disease and after various brain injuries [26–28]. Activation of microglia and astrocytes is closely associated with BBB dysfunction, and infiltration of plasma fibrinogen has been reported in Alzheimer disease [29] and other neurological conditions with BBB dysfunction [27, 31]. While the concentration of CSF fibrinogen has been reported to be low in PD patients [32], we found, for the first time, that there was no PD-associated infiltration of plasma fibrinogen in the SN, where dopamine neuron degeneration is an initial and specific pathology in PD. In contrast, a significant increase in fibrinogen infiltration was identified in the CN, where the neuronal degeneration was related to astrocytosis. Astrocytes play a role in angiogenesis [23] and inflammation [33]; both processes can cause BBB leakage. The relationship between astrocytosis and BBB dysfunction can be complex, as a beneficial role for astrocytes in removing fibrinogen has been suggested by in vitro studies [31]. Endothelial cell degeneration in the CN of PD cases has been suggested to represent age-related pathology [6]; thus, the disruption of BBB integrity in the CN may be a collective result of age and secondary pathology subsequent to initial neuronal degeneration in the SN. In contrast to the role of astrocytes, a contributing role for microglia in PD pathology, mainly neuronal degeneration, has been well described [33, 34].

The loss of neurons was found uniformly in all three brain regions examined. Degeneration of dopamine neurons has been well documented in the SN of PD patients [1]. Although there was loss of dopamine neurons in the SN, TH staining in the individual neurons was denser. This may be a compensatory response to the loss of dopamine neurons by increasing protein expression, as dopamine neurons are highly plastic in PD [1, 35]. Although degeneration of dopamine neurons in the SN is the most prominent pathology of PD, neuronal degeneration can affect other brain regions in advanced disease, for example the CN and cortical areas [3]. We also found neuronal degeneration in the CN and MFG brain regions of PD cases, as demonstrated by a reduction in neurons visualised by NeuN staining.

Atrophy of the CN in human PD cases has been described previously [36]. However, apart from changes related to degeneration of the dopamine system, for example reduced TH terminals, dopamine receptors and transporters, [37] our knowledge of neuronal degeneration in the CN is rather limited. NeuN is a widely used marker for mature neurons and labels Fox-3, a member of the RNA-binding protein family and regulator of neuronal mRNA splicing [38]. A low staining intensity of NeuN may also suggest a reduced expression of Fox-3. Neuronal degeneration in the frontal cortex is well established in PD cases, particularly those with dementia [39]. In our study, the extent of neuronal loss was similar in the CN and MFG; however, we only found elevation of astrocytes and leakage of the BBB in the CN, not in the MFG. Although both activation of astrocytes and dysfunction of the BBB may contribute to neuronal death, our data suggest that these processes are not the immediate cause of the neuronal degeneration seen in PD.

In conclusion, vascular degeneration in PD appears to be the result of endothelial cell degeneration, while retaining the BM. Increased string vessel formation may suggest a role for vascular hypoperfusion in the progression of PD. Leakage of the BBB may be associated with astrocytosis in the CN, which appears to be a pathological change secondary to the initial lesion in PD. The features of vascular degeneration in PD vary among different brain regions, as reflected by the differing extents of neuronal degeneration, astrocytosis, endothelial cell degeneration and dysfunction of the BBB in each region.

CONFLICT OF INTEREST

The authors have no conflict of interest to report.