A MORPHOLOGICAL AND IMUNOHISTOCHEMICAL STUDY OF THE

VASCULAR ABNORMALITIES IN NEUROPATHIC DIABETIC FOOT

Raluca Maria POPESCU1, Gina BOTNARIU1, Cristina Mihaela LACATUSU1, Bogdan
MIHAI1, Alina Delia POPA1, Eduard CATRINA2, Iulian BREZEAN2
1

UMF Gr.T.Popa Iasi

UMF Carol Davila Bucuresti

Abstract
Objectives: The aim of our study was to identify the changes who are important for the pathogeny and may
be for prognosis of diabetic foot. We studied 30 diabetic patients with a duration of diabetes between 14 and
20 years, all of them with diabetic neuropathy and plantar ulceration. We took bioptic fragments from the
ulcers edges which were specifically coloured, using current coloration (Hemalaun Eosin, van Gieson stain)
and special stains (PAS stain, trichromic C. Szekely stain).
Implication: Although the microangiopathy of the foot is not accepted as the primary cause for ulceration,
there are functional and morphological abnormalities of small vesels in diabetes. This aspects may be a
cornerstone for researchers as well as for practitioners in identification of pathogenic stages in development
of neuropathic ulceration and potential connection with microangiopathy as a starting point neural lesions.
Originality Value: The morphological changes shown in our study support the microangiopathic theory of
neuropathy without invalidating the metabolic one. Patients with diabetic neuropathy and plantar ulcerations
has vascular lesions affecting intima, but also the tunica media, causing endothelial hyperplasia, hialinosis
and mediocalcinosis in tunica media.
Keywords: neuropathic diabetic foot, microcirculation, arteriolar lesions, capillary lesions

INTRODUCTION
The importance of microvascular disease in the development of foot ulceration and in wound healing
has long been debated [1]. Although the microangiopathy of the foot is not accepted as the primary cause for
ulceration, there are functional and morphological abnormalities of small vesels in diabetes. Futhermore,
studies using physiological methods demonstrated functional microvascular abnormalities supporting the
involvement of microvascular disease in foot ulceration. From these observations, Parving put forward the
haemodynamic hypothesis to explain the development of microangiopathy in diabetes. According to this
hypothesis, in the early stages of diabetes there is blood flow disregulation leading to increased
microvascular flow and capillary pressure. This result in an injury response within the endothelial cells of the
microvasculature, consequently causing microvascular sclerosis [2; 3]. Nowadays it is known that there is a
close relation between the microcirculations anomalies and diabetic neuropathy [4; 5; 6], being
demonstrated that all the aspects of diabetic neuropathy (decrease of the nerve conduction velocity and of
the vibratory sensibility, alteration of the sudori-motor function or decrease of the muscle action potential) are
directly proportioned to the microvascular changes [7].
In the case of diabetic neuropathy we are dealing with a profound hemodynamic modification [8; 9],
which open the arterio-venous shunts, an abnormal adjustment of sanguine flow and also, an abnormal
inflammatory response to injury. The abnormal neurogenic adjustment of the microvascular hemodynamics
can contribute to the development of the microangiopathy, manifested by the thickening of the basement
membrane of the capilaries and proliferating lesions reported within the artery and arterioles lumens. Today it
is generally admitted that the microvascular abnormalities may contribute to the ischemic etiology of diabetic
neuropathy [10] and microvascular sclerosis leads to a structural locking of capillary wall, impairing
vasodilatation and disrupting other vascular responses particularly in the lower limbs. In diabetic neuropathy
there are abnormalities of the vasa vasorum, the microcirculation surrounding peripheral nerve fibers,
responsible for the diffusion of nutrients and also for clearance of waste products, is affected, resulting in
nerve damage [11]. This can be explain the development of diabetic foot ulceration but also, the impairment
of healing of the foot ulcers in neuropathic diabetic foot. However, until recently, only a few studies have
specifically examined the microcirculation in patients with foot ulceration to explore this relationship. Due to
the scarcity of descriptive information on the morphological changes in ulcerated neuropathic diabetic foot,
our present study aims to identify the morphological changes who are important for the pathogeny and may
be for prognosis of diabetic foot in terms of healing.

EXPERIMENTAL
We studied 30 diabetic patients (type 1 diabetes = 13 patients, 7 males and 5 females, type 2
diabetes = 17 patients, 9 males and 8 females) with a duration of diabetes between 14 and 20 years. All
patients had diabetic neuropathy and plantar ulceration. We considered eligible for this study only patients
with palpable pulses and ABI > 0.95 and <1.3. The patients needed minimal surgical treatment (only
superficial debridement, no minor/major amputation). We took bioptic fragments from the ulcers edges. (Cam
ce straturi ai urmarit sa iei- cat de groasa a fost biopsia?) These were specifically coloured, using current
coloration (Hemalaun Eosin, van Gieson stain) and special stains (PAS stain, trichromic C. Szekely stain).
Furthermore, we look after CD34, an imunohistochemichal marker that is used to specific highlight of
vascular endothelium.
Ce ai urmarit sa vezi?
RESULTS
The histopathological and imunohistochemical examination revealed specific microscopic changes
which fundament the pathogenic background of the particular lesions of the diabetic foot. In all cases studied
in optic microscopy were noticed arteriolar lesions that interested tunica intima and also media. They consist
in endothelial changes (swelling of endothelium and thickening of subendothelial layer (Figure 1),
appearance of lipid deposits in subendothelium (Figure 2)). We can notice even arteriole obstruction due to
the massive development of the intima throught the excessive proliferation of the subendothelial connective
tissue, followed, sometimes, by a fibrous change (Figure 3). There were also aspects of organized
thrombosis sometimes associated with the connective transformation of the thrombus (Figure 4).

Figure 1: Arteriola with endothelial cells with

a smoth inflated aspect, proliferation of the
subendothelial layer (van Gieson stain, ob 40x)

Figure 3: Intima with excesive proliferation of

the subendothelial layer, and the subsequent
obstruction of the lumen (HE stain ob 20x)

Figure 2: Arteriola with lipid deposits in

subendothelium (HE stain, ob 20x)

Figure 4: Thrombosis associated with connective

organization of the thrombus (HE stain ob 20x)

In the case of the large arterioles and arteries of muscular type we noticed the presence of the
fibrous tissue on the level of tunica media (Figure 5), and we also found aspects of mediocalcinosis or
mineralization at the limit of intima and tunica media (Figure 6) and calcium deposits on media level (Figure
7).

Figure 5: Arteriole with fibrous changes

(trichromic Szekely stain ob 10x)

Figure 6: Arteriole with subintimal mineralization

(HE stain ob 10x)

Figure 7: Mediocalcinosis limited to the media (HE stain ob 20x)

We used PAS technique to highlight the structures that contain carbohydrate elements (with vicinal
OH), like the glycoproteins and the glycosaminoglycans from the basement membranes structure. We
noticed that the basement membranes of microcirculation were thickened and PAS positive both in the deep
dermis, as well as in the superficial dermis (Figures 8). With the marker CD34 we found blood capillaries with
relative narrow lumens but also arterio-venous shunts. Quantitative ratio between capillary and arteriovenous shunts was different in the dermal papilla (Figure 9); it was also a different degree of expansion of
these structures lumens. In striate muscular tissue, where important dystrophic changes of the majority of
fibers were noticed (the edematous aspect of endomysium, till the replacement of degenerated muscular
tissue with adipose tissue), the immunoreactivity of CD34 was reduced to a few cappilaries, rarely present in
endomysium (Figure 10).

Figure 8: Deep dermis. Vessel group with

thickened basement membranes
(PAS Stain ob.20x)

Figure 9 Dermic papillas, capillaries and arteriovenous shunts. (CD34-positive immunoreactivity

ob. 20x)

Figure 10: Striate muscular tissue and

adipose tissue with reduced vascularization.
(CD34-positive immunoreactivity ob. 20x)

Figure 11: Peripheral nerve in striate muscular tissue

(CD34-positive immunoreactivity ob. 20x)

In peripheric nerves, in oblique section, a low CD34 immunomarking is noticed, indicating a

dimminuation of the number of endoneural capillaries. (Doar diminuarea numarului sau si posibil obstructia
lor- cred ca asta ar fi mai util pentru explicatia teoriei microvasculare a neuropatiei).
DISCUSSION
Abnormalities of the skin microcirculation are a feature of diabetes. Microcirculation has an important
role in skin nutrition, thus, it is not surprising that this has been linked to the development of diabetic foot
ulceration and with the impairment of healing of such a neuropathic foot ulcer. Structurally, the most notable
changes in the microcirculation involve thickening of basement membrane of the capillaries and proliferative
lesions noticed in the lumen of arteries and arterioles [2; 12]. However, the density of the skin capillaries
does not differ from healthy subjects [13]. The extent of basement membrane thickening has also been
observed to be related to the level of glycemic control, with increased basement thickening in poorly
controlled patients with diabetes [14]. The endothelium dysfunction plays an important part in the
pathogenesis of the diabetic microangiopathy [15-21]. It precedes the apparitions of the microvascular
lesions and it is proven by the decrease in the dilating response to the vasoactive agents and also through
alterations of the anti-thrombotic properties of the endothelium [18; 19; 22]. The sequence of events leading
to basement membrane thickening begins with the increased hydrostatic pressure and appearance of shear
forces in microcirculation. The precapillary efficient vasoconstriction capacity of foot is mainly reduced. This
fact causes an exposure of the capillary bed to a higher hydrostatic pressure. This is thought to evoke an
injury response on the part of the microvascular endothelium with subsequent release of extravascular matrix
proteins. All these produce an inflammatory response in vascular endothelium, causing later on a proteic
extravasation. In time, a more and more pronounced thickening of the basement membrane appears, and,
then, arteriolar hyalinosis [23]. Thickening of the basement membrane impairs the normal exchange of
nutrients and activated leukocyte migration between the capillary and interstitium. Furthermore, the elastic
properties of the capillary vessel wals are diminished, limiting their ability to vasodilate [24]. As a result, there
are produced reduction or loss of total hypaeremic response on every inflammatory process [15], a higher
arteriolar resistance and a greater circulation through arterio-venous shunts. Although thickening of
basement membrane does not produce a reduction of capillary lumen and microangiopathy cannot be
considered an oclusive microangiopathy, there is a deep hemodynamic modification [1; 24] which has an
important involvement in ischemic etiology of diabetic neuropathy. Functional alteration of microcirculation
can also explain the late cure noticed in pacients with diabetes mellitus [25], also a greater susceptibility to
infections in diabetic foot [16]. Functional ischemia is expressed by an impairement of microcirculation
function of responding through vasodilation to several injuries [26].
. This is due to blood shunting from nutritive capillaries through arterio-venous shunts, which have a lower
resistance compared to capillary vessels. These shunts have a sympathetic innervation, so, the presence of
vegetative diabetic neuropathy with concomitant sympathetic denervation may lead to their opening,
resulting a faulty distribution of blood flow between nutritive capillaries and underpapillary vessels. This leads
to a reduction of capillary blood flow, to development of peripheral diabetic neuropathy and other
complications of neuropathic diabetic foot [1; 13; 26]. Microcirculation modifications, similar to the ones
described in skin, were noticed in striate muscles too [14]. These modifications explain the presence of
degenerative lesions in skeletal muscle fibers, in the absence of changes with inflammatory attributes.
Aspectele histopatologice evidentiate arata ca la pacienti cu neuropatie si leziuni ulcerative neuropate,
exista modificari microvasculare atat la nivel cutanat cat si muscular (asta poate insemna ca modificarile
vasculare nu sunt date de leziune, presiune, forfecare, infectie) desi pacientii nu sunt vasculopati dupa

criteriile ABI. Asta poate sustine cateva aspecte:

- leziunile microvasculare gasite ar putea fi prima modificare aparuta si ar putea fi prezente si la nivelul vasa
nervorum, ceea ce ar putea sustine teoria microangiopatica a neuropatiei diabetice. (ar trebui explorate
modificarile la nivelul vasa nervorum)
- leziunile microvasculare existente sunt urmarea neuropatiei diabetice (prin deschiderea sunturilor,
alterarea tonusului simpatic) si se pune problema daca ele sunt doar focalizate intr-o anumita regiune
(acolo unde apare ulceratia prin supraadaugarea factorului presiune necontrolata) sau sunt difuze. pentru
a putea lamuri aceasta problema un studiu urmator ar trebui sa compare leziunile gasite din regiunea
ulceratiei si din alta regiune plantara (una presionala, alta fara presiune) dar biopsia profunda din regiuni
sanataoase poaye pune probleme de etica. daca sunt localizate este posibil ca leziunile vascualre sa fie
efectul presiunii determinate de neuropatie.daca sunt difuze atunci leziunile vasculare ar putea fi
consecinta neuropatiei diabetice!!!!
CONCLUSIONS
The morphological changes shown in our study may support the microangiopathic theory of neuropathy
without invalidating the metabolic one. Patients with diabetic neuropathy and plantar ulcerations has vascular
lesions affecting intima, but also the tunica media, causing endothelial hyperplasia, hialinosis and
mediocalcinosis in tunica media. This can explain the development of diabetic foot ulceration but also, the
impairment of healing of the foot ulcers in neuropathic diabetic foot.
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Corresponding author
Botnariu Gina Eosefina, lecturer,
Bd Independentei 1, Iasi,
ginabotnariu66@gmail.com