the age-related morphological changes in the capillary

1

The Age-Related Morphological Changes in the Capillary Architecture of

the Peripheral Nerve in Rats

MASAHIRO SAKITA

Abstract.[Purpose] The impairment of the peripheral nerve with aging is associated with capillary degradation and apoptosis. The purpose of this study was to morphologically investigate whether the capillary architecture in the tibial nerve spontaneously declines with age in rats. [Subjects] Male Sprague-Dawley rats (n=10) were used in the present study. [Methods] The rats were ran-domly divided into control (n=5), elderly (n=5) groups. The body mass, adipose weight, capillary diameter, cross-sectional area and number of microvascular ramifications in the tibial nerve were compared between the control (56-weeks-old) and elderly (106-weeks-old) groups using the three-dimensional images captured by confocal laser scanning microscopy. [Results] The body mass and adipose weights in the elderly group were significantly higher than those ob-served in the control group (p < 0.05). Conversely, the capillary diameter, cross-sectional area and number of microvascular ramifications in the elderly group were significantly lower than those observed in the control group (p < 0.05). [Conclusion] These findings showed that the capillaries in the tibial nerve mor-phologically degrade with age. Such capillary degradation with age may be associated with an increase in the adipose weight which thus causes lipid peroxidation and cell apoptosis.Key words: capillary in the tibial nerve, aging, three-dimensional image

2

1. INTRODUCTION

Functional deficits of the peripheral nerve in the lower extremities have been linked to aging. The neural dysfunction with aging is caused by structural, biochemical and molecular biological changes that gradually progress to nerve degradation. Previous studies have shown that functional deficits of the periph-eral nerves were related to the occurrence of falling in the elderly (Overstall et al., 1977; Tinetti et al., 1988). The age-related subclinical peripheral neu-ropathy is greatly influenced by the degradation of the microvessels. The structural alterations of the microvessels supplying glucose and oxygen to peripheral nerves result in neuropathy (Girach and Vignati, 2006). In terms of the cerebral microvascular pathology, the microvascular abnormality compli-cates or precedes neurodegeneration (van Dijk et al., 2008; Brown, 2009). Therefore, it is assumed that maintenance of the microvascular structure and physiological function can help to prevent the peripheral nerve degradation. The metabolism of peripheral nerves includes both intracytoplasmic glycolysis and intramitochondrial oxidative phosphorylation processes that produce ade-nosine triphosphate (ATP) and creatine phosphate (Verdú et al., 2000). The amounts of oxygen and glucose transported via microvessels used for the en-doneural oxidative glycolysis process are decreased by the reduction of periph-eral nerve blood flow in aged rats (Low et al., 1986).

Conversely, angiogenesis is promoted by VEGF (vascular endothelial growth factor), which is a growth factor for endothelial cells, and VEGF plays a role in capillary sprouting and arterial differentiation (Ogunshola et al., 2002; Mukouyama et al., 2005). VEGFs are elicited in response to hypoxic conditions through hypoxia inducible factor-1 (HIF-1) which is a transcription factor (Saint-Geniez and D’Amore, 2004). The apoptosis cascade of the capil-laries can be induced when a decrease or loss of VEGF occurs (LaManna et al., 2004).

The Age-Related Morphological Changes in the Capillary Architecture of the Peripheral Nerve in Rats

3

The decline of VEGF expression in cerebral microvessels appears to occur following the loss of the reactivity of HIF-1 to hypoxia with aging (Chavez and LaManna, 2003; Rivard et al., 2000). HIF-1 is stored in the neuronal cyto-plasm, and its accumulation can lead to the inhibition of HIF-1 transcriptional activity in the old rat cerebral cortex (Abe and Berk, 2002; Rapino et al., 2005). The decline in HIF-1 transcriptional activity with aging is associated with neuronal death via the resulting downregulation of VEGF expression. In addition, the dysregulation of mitochondrial biogenesis led to metabolic hy-poxia in animal experiments (Addabbo et al., 2009). The mitochondrial dys-function appears to contribute to an imbalance in the cellular energetic me-tabolism, oxidative stress and endothelial dysfunction, all of which are associated with aging (Gutierrez et al., 2006). The impairment of mitochondria with aging is likely to promote the production of reactive oxygen species (ROS), and ROS can damage the DNA of endothelial cells and elicit microvas-cular degradation with aging (Ungvari et al., 2007). Therefore, the failure of angiogenesis induced by hypoxia seems to be caused by the decrease in mito-chondrial oxidizing enzyme activity, HIF-1 transcriptional activity and VEGF expression.

However, it is not morphologically clear whether the degradation of the capillaries in the peripheral nerves spontaneously occurs with aging, although the mechanisms of age-related microvessel degradation are gradually being uncovered. Therefore, we investigated the morphological changes of the capil-lary architecture in the peripheral nerves during the spontaneous aging of rats.

2. MATERIALS AND METHODS

2.1 Animals.The present study was approved by the Animal Care and Use Committee of Himeji Dokkyo University and was performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Na-

4

tional Research Council, 1996). Male Sprague-Dawley (SD) rats (10 weeks old, n=10) were used in this study, and were chosen at random for use in the con-trol (n=5) and elderly (n=5) groups at 20 weeks old. The rats were housed for 46 (control group) and 96 (elderly group) weeks in a room maintained on a 12-hour light and dark cycle at a temperature of 22 ± 2°C with 40–60% humidity. These rats were all housed in cages of the same size, and food and water were provided ad libitum.

2.2 Nerve sample preparation.The body masses of the rats were measured until they were 56 (control) and 106 (elderly) weeks old. Each rat was then anaesthetized with pentobarbital sodium (50 mg/kg; i.p.). The abdominal cavity was opened and both the left common iliac artery and vein were ligated and extraction from the testicular fat. Then a catheter was inserted to perfuse the abdominal aorta with contrast medium to the right hind limb. A series of nerves, from the sciatic nerve to the distal end of the tibia in the right hind limb was perfused for 3 min to wash out the intravascular blood with a 0.9% physiological saline solution containing 10,000 IU/L heparin at 37°C, 10% glucose and the contrast medium. The con-trast medium was composed of 1% fluorescent material (PUSR80; Mitsubishi Pencil, Tokyo, Japan), 8% gelatin (Nakalai Tesque, Kyoto, Japan) and distilled water was administered into the circulation to a series of nerves from the sciatic nerve to the distal end of the tibia. The whole body of the rat was then immersed in cold saline for 10 min after the perfusion with contrast medium. The series of nerves were then excised and frozen in isopentane precooled in liquid nitrogen. The nerves were ultimately stored at -80°C (Murakami et al., 2010).

2.3 Three-dimensional (3-D) visualization of the capillary architecture.To observe the 3-D architecture of the capillary, confocal laser scanning mi-croscopy (CLSM) (TCS-SP; Leica Instruments, Mannheim, Germany) was used


5

in the fluorescent mode with an argon laser (488 nm) (Toyota et al., 2002; Fujino et al., 2005). The nerve sample was cut at a distance 5 mm from the distal end of the nerve using a cryostat microtome (CM3050; Leica Microsys-tems, Mannheim, Germany) at -20°C, and then the cut sample block was fixed with 4% paraformaldehyde (pH 7.4) and thawed to room temperature for 10 min. Images from the CLSM were visualized using a ×20 objective lens. Each sample block that was perfused with contrast medium was scanned and visual-ized to a depth of 50 μm at 1 μm/slice, and 3-D images were visualized with a depth of 50 μm. From the 3-D images that were made by the CLSM, the capil-lary diameter, cross-sectional area and number of microvascular ramifications per sample block from 40–60 capillaries were measured in a few images of a 800 × 800 μm2 area using the ImageJ software program.

2.4 Statistical analysis.A Mann-Mann-Whitney’s U test was performed to compare the body mass between the control and elderly groups. An unpaired Student’s t-test was per-formed to compare the various factors, including the capillary diameter, adi-pose weight (the testicular fat weight), cross-sectional area and the number of microvascular ramifications between the control and elderly groups. Differ-ences in P-values of less than 0.05 were considered to be statistically significant (p < 0.05).

3. RESULTS

3.1 Body mass and adipose weight.Mann-Mann-Whitney’s U test and Student’s t-test revealed that there were significant differences between the control and elderly groups (Table 1). Both the body mass and adipose weight in the control group were significantly higher than those in the elderly group (p < 0.05).

6

3.2 Capillary diameter, cross-sectional area and number of microvascular ramifications.

The capillary diameter of the control rats was 7.63 ± 0.11μm and that of the elderly rats was 4.86 ± 0.12μm. The capillary cross-sectional areas of the con-trol and elderly rats were 52.90 ± 1.47 × 10-3mm2 and 21.24 ± 0.93 × 10-3mm2, respectively. The number of microvascular ramifications of the control and elderly rats were 47.13 ± 1.82 and 30.88 ± 4.44, respectively. The mean values of the capillary diameter, capillary cross-sectional area and the number of mi-crovascular ramifications were significantly (p < 0.05) smaller in the elderly group than in the control group, as determined by Student’s t-test (Figure 1).

Table 1. The body mass and adipose weight of rats in Control and Elderly groups

Cont (56 weeks old) Elder (106 weeks old)

Body mass (g) 616.0 ± 22.8 953.5 ± 120.0*

Adipose weight (mg) 5163.1 ± 346.9 9429.1 ± 1037.1*Control and Elderly groups were presented Cont and Elder groups, respectively. Mann-Whitney’s U test showed that the body mass was different between Cont and Elder groups (p<0.05). Unpaired Student’s t - test showed that the body mass and adipose weight were different between Cont and Elder groups (p<0.05). The values of the body mass are presented as median ± quartile deviation.The values of the adipose weight are presented as the mean ± SEM. *: significant difference between the Cont and Elder groups (p<0.05)

Figure 1. Unpaired Student’s t-tests showed that the mean capillary diameter (A), cross-sectional area (B) and number of microvascuar ramifications (C) were significantly differ-ent between the two groups (p<0.05). The values are presented as the means ± SEM. *: Significant difference between the Control and Elderly groups determined by the un-paired Student’s t-test (p<0.05)


7

3.3 Capillary architecture constructed by CLSM.The capillaries in both the control and elderly groups were indicated by the 3-D images constructed by the CLSM (Figure 2). The capillaries in the tibial nerves of the control group were located close together and had anastomoses. Conversely, the 3-D image of the capillaries in the elderly group showed that there were empty spaces caused by the capillary loss and degradation.

4. DISCUSSION

We morphologically investigated that age-related decline of the capillaries in the tibial nerves of rats in this study. Our findings obtained by analyzing the capil-laries of the tibial nerves using the 3-D images captured by CLSM are the first evidence that shows the capillary regression in the peripheral nerves with aging.

The body mass was increased in the elderly group compared to the control group. The adipose weight in the elderly group was also increased compared with that in the control rats. These findings indicate that the age-related in-

Figure 2. Confocal laser scanning microscopic images of the capillaries in the tibial nerves of rats. In both images, the tibial nerve runs from the upper right to the lower left. The capillaries in the Elderly group appeared to be less dense compared with those in the Control. The arrows indicate the capillaries. Scale bar = 100 μm.

8

crease in the body mass is associated with an increase in adipose tissue (Shok-ouhi et al., 2008). Conversely, the capillary diameter, cross-sectional area and the number of microvascular ramifications in the elderly rats were decreased compared to those in the control rats. These declines could mean that the degradation and/or the apoptosis of the capillaries in the tibial nerves occurs progressively with aging.

The major finding of recent studies is that cell apoptosis was associated with elevated intraneuronal levels of reactive oxygen species (ROS) (Thra-sivoulou et al., 2006; Finkel and Holbrock, 2000; Wickens, 2001) and free radicals (Wickens, 2001). A decrease in the reactivity of HIF-1, which is a transcription factor promoting angiogenesis, gradually occurs with aging (Abe and Berk, 2002; Rapino et al., 2005). The increase in oxidative stress, such as ROS and free radicals, may indirectly cause capillary apoptosis. In the cerebral microvessels, the presence of intracellular hypoxia induces the decrease in the reactivity of HIF-1, and this leads to a subsequent decrease in VEGF expres-sion (Chavez and LaManna, 2003; Rivard et al., 2000). In brief, this decrease in VEGF expression can eventually lead to a decrease in the capillary diame-ter, cross-sectional area and the number of microvascular ramifications in the elderly rats.

In our pilot study, the capillary diameter, cross-sectional area and the num-ber of microvascular ramifications in aged rats that performed aerobic exer-cise for ten weeks were increase compared to those in the non-exercising rats (Sakita et al., in press). It has been shown that aging is associated with an in-creased level of nerve malondialdehyde (a marker of lipid peroxidation) and a higher level of Schwann cell apoptosis in sedentary rats (Shokouhi et al., 2008). However, the nerve lipid peroxidation and Schwann cell apoptosis were dimin-ished in the rats that exercised. The lipid peroxidation of both Schwann cells and the capillaries can lead to the formation of free radicals, which can injure the nerve tissue and endothelial cells (Ji and Fu, 1992; Bejma and Ji, 1999). Furthermore, the mitochondrial dysfunction associated with aging seems to


9

produce ROS, and these ROS may lead to the microvascular degradation that occurs with aging (Ungvari et al., 2007).

It has been demonstrated that aerobic training increased the levels of suc-cinate dehydrogenase (SDH) activity, which is an indicator of the mitochon-drial oxidative enzyme activity, in the skeletal muscle of rat. The SDH activity could lead to angiogenesis (Wüst et al., 2009; Murakami et al., 2010). The mitochondrial oxidative enzyme activity in neurons and capillaries should pro-mote lipid metabolism and decreased free radicals and ROS. Therefore, the preservation or restoration of the capillaries in the peripheral nerves may be linked to mitochondrial oxidative enzyme activity, the decrease in free radicals and improvement of the HIF-1 response to hypoxia. Thus, the VEGF expres-sion will ultimately be upregulated, and the capillary sprouting may be reacti-vated throughout the aerobic exercise.

The present study has morphologically demonstrated that spontaneous ag-ing elicited the capillary degradation and loss of tibial nerves. However, it was not shown whether the oxidative stress and ROS increase or whether the HIF-1 and VEGF expression decrease with spontaneous aging. Therefore, future studies should immunohistochemically and molecular biologically research the expression levels of both these negative and positive factors and their relation-ship with the peripheral nerve dysfunction associated with aging.

AcknowledgementThe present study was supported by Grants-in-Aid for the Academic Encouragement of researchers (No. 12001) from Kyoto Tachibana University.

REFERENCESAbe J, Berk BC (2002). Hypoxia and HIF-1alpha stability: another stress-sensing mecha-

nism for Shc. Circ Res. 91: 4–6.Addabbo F, Ratliff B, Park HC, Kuo MC, Ungvari Z, Csiszar A, Krasnikov B, Sodhi K, Zhang

F, Nasjletti A, Goligorsky MS (2009). The Krebs cycle and mitochondrial mass are early victims of endothelial dysfunction: proteomic approach. Am J Pathol. 174: 34–43.

10

Bejma J, Ji LL (1999). Aging and acute exercise enhance free radical generation in rat skeletal muscle. J Appl Physiol. 87: 465–470.

Brown WR, Moody DM, Thore CR, Anstrom JA, Challa VR (2009). Microvascular chang-es in the white mater in dementia. J Neurol Sci. 283: 28–31.

Chavez JC, LaManna JC (2003). Hypoxia-inducible factor-1alpha accumulation in the rat brain in response to hypoxia and ischemia is attenuated during aging. Adv Exp Med Biol. 510: 337–341.

Finkel T, Holbrock NJ (2000). Oxidants, oxidative stress and the biology of ageing. Na-ture. 408: 239–247.

Fujino H, Kohzuki H, Takeda, Kiyooka T, Miyasaka T, Mohri S, Shimizu J and Kajiya F (2005). Regression of capillary network in atrophied soleus muscle induced by hindlimb unweighting. J Appl Physiol. 98: 1407–1413.

Girach A , Vignati L (2006). Diabetic microvascular complications-can the presence of one predict the development of another? J Diab Complic. 20: 228–237.

Gutierrez J, Ballinger SW, Darley-Usmar VM, Landar A (2006). Review Free radicals, mitochondria, and oxidized lipids: the emerging role in signal transduction in vascu-lar cells. Circ Res. 99: 924–932.

Ji LL, Fu RG (1992). Responses of glutathione system and antioxidant enzymes to exhaus-tive exercise and hydroperoxide. J Appl Physiol. 72: 549–554.

LaManna JC, Chavez JC, Pichiule P (2004). Structural and functional adaptation to hy-poxia in the rat brain. J Exp Biol. 207: 3163–3169.

Low PA, Schmelzer JD, Ward KK (1986). The effect of age on energy metabolism and resistance to ischemic conduction failure in rat peripheral nerve. J Physiol. 374: 263–271.

Mukouyama YS, Gerber HP, Ferrara N, Gu C, Anderson DJ (2005). Peripheral nerve-de-rived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feed-back. Development. 132: 941–952.

Murakami S, Fujino H, Takeda I, Momota R, Kumagishi K, Ohtsuka A (2010). Comparison of capillary architecture between slow and fast muscles in rats using a confocal laser scanning microscope. Acta Med Okayama. 64: 11–18.

Ogunshola OO, Antic A, Donoghue MJ, Fan SY, Kim H, Stewart WB, Madri JA, Ment LR (2002). Paracrine and autocrine functions of neuronal vascular endothelial growth factor (VEGF) in the central nervous system. J Biol Chem. 277: 11410–11415.

Overstall PW, Exton-Smith AN, Imms FJ, Johnson AL (1977). Falls in the elderly related


11

to postural imbalance. Br Med J. 1: 261–264.Rapino C, Bianchi G, Di Giulio C, Centurione L, Cacchio M, Antonucci A, Cataldi A (2005).

HIF-1alpha cytoplasmic accumulation is associated with cell death in old rat cerebral cortex exposed to intermittent hypoxia. Aging Cell. 4: 177–185.

Rivard A, Berthou-Soulie L, Principe N, Kearney M, Curry C, Branellec D, Semenza GL, Isner JM (2000). Age-dependent defect in vascular endothelial growth factor expres-sion is associated with reduced hypoxia-inducible factor 1 activity. J Biol Chem. 275: 29643–29647.

Saint-Geniez M, D’Amore PA (2004). Development and pathology of the hyaloid, choroidal and retinal vasculature. Int J Dev Biol. 48: 1045–1058.

Sakita M, Murakami S, Fujino H. The morphological changes in the capillary architecture of the tibial nerve associated with spontaneous aging and aerobic exercise interven-tion during aging in rats. J Phys Ther Sci. (in press)

Shokouhi G, Tubbs RS, Shoja MM, Roshangar L, Mesgari M, Ghorbanihaghjo A, Ahmadi N, Sheikhzadeh F, Rad JS (2008). The effects of aerobic exercise training on the age-related lipid peroxidation, Schwann cell apoptosis and ultrastructural changes in the sciatic nerve of rats. Life Sci. 82: 840–846.

Thrasivoulou C, Soubeyre V, Ridha H, Giuliani D, Giaroni C, Michael GJ, Saffrey MJ, Cowen T (2006). Reactive oxygen species, dietary restriction and neurotrophic fac-tors in age-related loss of myenteric neurons. Aging Cell. 5: 247–257.

Tinetti ME., Speechley M, Ginter SF (1988). Risk factors for falls among elderly persons living in the community. N Engl J Med. 319: 1701–1707.

Toyota E, Fujimoto K, Ogasawara Y, Kajita T, Shigeto F, Matsumoto T, Goto M, Kajiya F (2002). Dynamic changes in three-dimensional architecture and vascular volume of transmural coronary microvasculature between diastolic- and systolic-arrested rat hearts. Circulation. 105: 621–626.

Ungvari Z, Orosz Z, Labinskyy N, Rivera A, Xiangmin Z, Smith K, Csiszar A (2007). In-creased mitochondrial H2O2 production promotes endothelial NF-kappaB activation in aged rat arteries. Am J Physiol Heart Circ Physiol. 293: H37–H47.

van Dijk EJ, Prins ND, Vrooman HA, Hofman A, Koudstaal PJ, Breteler MM (2008). Progression of cerebral small vessel disease in relation to risk factors and cognitive consequences: Rotterdam Scan study. Stroke. 39: 2712–2719.

Verdú E, Ceballos D, Vilches JJ, Navarro X (2000). Influence of aging on peripheral nerve function and regeneration. J Peripher Nerv Syst. 5: 191–208.

12

Wickens AP (2001). Ageing and the free radical theory. Resp Physiol. 128: 379–391. Wüst RCI, Gibbings SL, Degens H (2009). Fiber capillary supply related to fiber size and

oxidative capacity in human and rat skeletal muscle. Adv Exp Med Biol. 645: 75–80.

the age-related morphological changes in the capillary

Documents