Biomedical subjects

D N Angelov

Publications and source records attributed to D N Angelov.

At least 37 records · Page 2Linked to original sources

PubMed1998

[Nerve-plasty interventions on the facial nerve in the elderly patient--morphological evaluation of disappointing functional results].

BACKGROUND: Nerve sutures such as facial-facial anastomosis are helpful methods for recovery of facial movements after peripheral nerve lesions. Most of the results on neuronal regeneration were based on experiments with young or young adult animals. In contrary the vast majority of the diseases (cholesteatoma or tumors of the parotid gland) causing facial nerve lesions are in aged patients. METHODS: Therefore, we compared the original data from two recently published articles concerning the axonal outgrowing process after facial-facial-anastomosis in young-adult and aged rats. In additional, we tried to explain the clinically observed postparalytic syndrome (synkinesia, autoparalytic syndrome,...) after surgical interventions on the facial nerve with the experimental results in the rat. RESULTS: As an important result, we could not find spontaneous loss of facial motoneurons on the control side in the aged rats. On the operated side, two results have to be emphasized. The initial regeneration (10-42 days after the operation) showed a significant faster reinnervation in the group of young rats. The aged rats showed an apparent hyperinnervation after axonal outgrow to the mimic muscles has been accomplished. CONCLUSION: Both experimental observations are in agreement with our clinical experiences. After facial nerve surgery, aged patients show a delayed recovery of the mimic functions and a more pronounced postparalytic syndrome. A morphological explanation one can bear is that the delayed reinnervation causes the extreme hyerinnervation, which leads to a simultaneous innervation of different muscles by same facial motoneurons.

Adult↗

PubMed1998

Significance of trigeminal sensory input on regrowth of hypoglossal and facial motoneurons after hypoglossal facial anastomosis in rats.

Hypoglossal facial anastomosis (HFA) is a standard surgical technique for restoration of facial movements in cases of intratemporal lesions of the facial nerve. Case reports provide evidence that an affected trigeminal system reduces functional outcome. In order to detect morphological changes in the hypoglossal nucleus responsible for this phenomenon, we used 18 Wistar rats and performed three different surgical combinations. In group 1, six animals received HFA only. In group 2, HFA was combined with resection of the contralateral infraorbital nerve. In group 3, HFA was combined with resection of the ipsilateral infraorbital nerve. Fifty-six days after the operation, horseradish peroxidase (HRP) was injected into the whisker pad. As shown in previous studies using HRP, retrograde-labelled motoneurons occurred in the hypoglossal and facial nuclei. Counts of the labelled motoneurons showed no change in the number of projecting hypoglossal motoneurons in group 2 when compared to HFA only, but a significantly smaller number in group 3 (-35%). Furthermore, the number of projecting facial motoneurons was significantly reduced in group 2 (-85%) and group 3 (-45%). These morphological findings indicate an absent or insufficient functional connection between the contralateral infraorbital nerve and the hypoglossal nucleus, and a strong influence of the infraorbital nerve to the ipsi- and contralateral facial nuclei. Additionally, our study provides morphological evidence that the integrity of the sensory trigeminal system is very important in reconstructive facial nerve surgery.

Analysis of Variance↗

PubMed1998

Temporospatial relationships between macroglia and microglia during in vitro differentiation of murine stem cells.

Embryonic stem (ES) cells of the permanent line BLC6 derived from a 129/Sv Gat mouse blastocyst were differentiated as spheroid aggregates (embryoid bodies, EBs) in the presence of retionic acid. After 2 days in suspension, EBs were plated on gelatine-coated glass coverslips and cultivated for 5, 9, and 16 days post plating (DPP) in normal medium. In this study we investigated whether the well-known retinoic acid-induced differentiation of ES cells into neurons (identified by immunostaining for neuron-specific enolase and synaptophysin) was accompanied by cells expressing astroglial (GFAP), oligodendroglial (O4), and microglial (5C6, galectin-3) markers. Whereas differentiation of neurons was closely related to their centrifugal migration towards the periphery of the EBs, the maturation of neuroglia followed a strict time-dependent manner. At 5 DPP, only neurons but no cells expressing glia-specific markers, were observed. At 9 DPP, GFAP-positive and O4-positive macroglial cells appeared. At 16 DPP, microglial cells (5C6-positive and galectin-3-positive) occurred. The established dynamic of relationships between neuronal and nonneuronal cells shows that the model of EBs is similar to the sequence differentiation of the nervous tissue. Thus, enabling in vivo observation of neurons, astrocytes, oligodendrocytes, and microglia, the model of EBs provides a basis for further investigations on the relationships between neurons and neuroglia under various experimental conditions.

Animals↗

PubMed1998

Altered expression of immune-related antigens by neuronophages does not improve neuronal survival after severe lesion of the facial nerve in rats.

Injection of Fluoro-Gold (FG) into the whiskerpad muscles of rats yields a permanent retrograde labeling of motoneurons in the facial nucleus. Following subsequent resection of 10 mm of the facial nerve, one-third of the facial motoneurons die and the microglia phagocytize the dead FG-labeled neurons, take up FG, and get labeled in vivo. The resulting identification of all FG-labeled cells allows long-term comparative investigations on the behavior of neuronophages. In this study, we used two groups of rats to test whether the quantified expression of five immune-related antigens by neuronophages was related to quantified decline in neuron number (counts after immunostaining for neuron-specific enolase) 3 to 224 days after resection of the facial nerve. Rats of the first group received standard food and those of the second group, pellets containing 1,000 ppm of the calcium channel blocker nimodipine. Image analysis of the number of FG-containing cells and the number and projection area of immunopositive neuronophages in serial sections for each antigen showed that nimodipine significantly attenuated the immunostaining for CR3, MHC class I, and class II antigens (monoclonal antibodies [MAbs] OX-42, OX-18, and OX-6); enhanced the expression of monocyte-macrophage-specific antigen (MAb ED1); and did not change the expression of rat macrophage differentiation antigen (MAb ED2). The altered expressions, however, had no effect on the loss of motoneurons in the lesioned facial nucleus. We conclude that the degree of expression of immune-related antigens by neuronophages has no influence on the delayed neuronal cell death induced by permanent target deprivation.

Animals↗

PubMed1997

Delayed hypoglossal-facial nerve suture after predegeneration of the peripheral facial nerve stump improves the innervation of mimetic musculature by hypoglossal motoneurons.

Surgical reconstruction of the facial nerve is common clinical practice following destruction of the intracranial facial nerve. Delayed hypoglossal-facial anastomosis (HFA) is the procedure of choice, although the effect of delay on outcome remains unclear. To study the effect of delayed anastomosis on reinnervation, we sutured the proximal stump of a freshly transected hypoglossal nerve of Wistar rats to the distal stump of the ipsilateral facial nerve, which had been transected 7-56 days earlier. Animals that had received HFA without delay served as the control group. Forty days after HFA, horseradish peroxidase (HRP) was injected into the whisker pad; 2 days later, the animals were killed. Reinnervation was assessed by determining the proportion of labeled neuronal cell bodies in the brainstem. The control group had 68% reinnervation of these muscles by hypoglossal neurons and had 32% reinnervation by facial neurons. When the distal facial nerve had been allowed to degenerate for 7 days before HFA, reinnervation of the hypoglossal nerve decreased to 54%, and reinnervation by the facial nerve increased to 46%. However, after a delay of 10-56 days, the hypoglossal fraction increased and stabilized at 77%, and the facial motoneuron fraction decreased to 23%. The presence of new neuromuscular junctions was confirmed by HRP labeling of motor end plates in vivo and by electromyography. We conclude that, under the conditions of hypoglossal-facial crossed nerve suture, the predegeneration of the distal stump of a transected facial nerve enhances the reinnervation of facial muscles by hypoglossal axonal sprouts.

Animals↗

PubMed1997

Nimodipine maintains in vivo the increase in GFAP and enhances the astroglial ensheathment of surviving motoneurons in the rat following permanent target deprivation.

Facial and hypoglossal nerves were resected unilaterally in a total of 108 rats. Rats were divided into two groups; one group received standard food pellets (placebo), the other received food pellets containing the Ca(2+)-blocking agent nimodipine. The expression of glial fibrillary acidic protein was examined in paraffin sections of the brainstem using light microscopical immunocytochemistry, and the degree of glial process ensheathment of the surviving neuronal perikarya in the hypoglossal and facial nuclei quantified on electron micrographs. Up to 28 days post-axotomy no differences in glial fibrillary acidic protein-immunoreactivity were observed between placebo and nimodipine-treated animals. By 42-56 days, glial fibrillary acid protein-immunoreactivity was stronger in the nimodipine treated animals and by 112 days, glial fibrillary acid protein-immunoreactive astrocytes occurred only in nimodipine-treated animals. Thin astrocytic processes were seen to ensheath neurons in both placebo and nimodipine-treated animals. By 28 days post axotomy, lesioned neurons in nimodipine treated animals were covered by a mean of 2.6 (hypoglossal) and 2.9 (facial nucleus) astrocytic lamellae, compared with 1.7 lamellae in the placebo group. This relatively greater ensheathment of hypoglossal and facial neurons was maintained up to 112 days post-lesion, but reduced in the placebo-treated group to approximately 1.4 lamellae. It is concluded that nimodipine enhances the formation of astrocytic lamellae on lesioned neurons and that this process may be associated with a protective role for activated astrocytes directed towards motoneurons suffering from permanent target-deprivation.

Animals↗

PubMed1997

Effect of delayed facial-facial nerve suture on facial nerve regeneration. A horseradish peroxidase tracing study in the rat.

In clinical practice, the lesioned facial nerve is usually restored by facial-facial nerve anastomosis (FFA) with some delay. The optimal time-point for facial nerve reconstruction is still unknown. This study, using rats, compared the effects of immediate and delayed FFA, i.e. FFA 7-56 days after interruption of the facial nerve. Muscle reinnervation was studied 42 days after nerve suture by counting all retrogradely labelled facial motoneurons after injection of horseradish peroxidase (HRP) into the whiskerpad of the rats. Immediate FFA caused a local hyperinnervation of the target muscle, i.e. the projection of more neurons into the whiskerpad muscles than under normal conditions. FFA delayed for 7 days resulted in a significant suppression of this hyperinnervation, whereas longer delay times of 10-56 days showed no difference from immediate FFA.

Analysis of Variance↗

PubMed1996

Nimodipine accelerates axonal sprouting after surgical repair of rat facial nerve.

Facial-facial anastomosis (FFA), i.e., suture of transected facial nerve, was performed in adult Wistar rats. For 10-112 d post-operation (DPO), half of the animals received standard food (placebo) and half received food pellets containing 1000 ppm nimodipine, a Ca2+ channel blocker. The time course of mimetic reinnervation between these two groups was compared by counting all retrogradely labeled motoneurons after injection of horseradish peroxidase (HRP) into the whiskerpad. In unoperated animals, injection of HRP labeled 1280 +/- 113 motoneurons. After FFA, this number dropped to zero, and the first HRP-labeled facial motoneurons reappeared in both placebo- and nimodipine-treated animals at 14 DPO. The treatment with nimodipine yielded two beneficial effects. (1) It accelerated axonal sprouting until 28 DPO. Whereas the number of HRP-labeled cells in the placebo group was 171 +/- 9 (mean +/- SD) at 16 DPO, 372 +/- 43 at 21 DPO, and 636 +/- 187 at 28 DPO, the number of sprouted motoneurons in nimodipine-treated rats was twice as high: 386 +/- 34 at 16 DPO, 620 +/- 28 at 21 DPO, and 756 +/- 257 at 28 DPO. (2) Nimodipine reduced the polyneuronal innervation of the target muscles. Whereas the number of HRP-labeled cells in the placebo group increased to 1430 +/- 36 at 56 DPO and 1600 +/- 31 at 112 DPO, the number of labeled motoneurons in nimodipine-treated rats remained almost within the normal range: 1315 +/- 31 at 56 DPO and 1354 +/- 33 at 112 DPO.

Anastomosis, Surgical↗

PubMed1996

ED2-positive perivascular cells act as neuronophages during delayed neuronal loss in the facial nucleus of the rat.

Injection of Fluoro-Gold (FG) into the whisker pad of rats yields a stable retrograde labeling of facial motoneurons. After removal of 10 mm from the facial nerve the microglia phagocytose the FG-prelabeled dead neurons and assume the label. A subsequent brightfield immunostaining of the sections with HRP-DAB as end-product fully quenches the fluorescence of FG from all specifically stained structures (immunoquenching). Combining FG-labeling of neuronophages with immunoquenching, we recently described a population of enigmatic fluorescent cells, found in immediate vicinity to the motoneurons after the general neuronofugal migration of microglia. As the fluorescence of these cells was not quenched after a triple immunostaining with anti neuron-specific enolase, anti-GFAP, and OX-42 (quenching all fluorescence from neurons and glia), they seemed to represent a new, immunologically not identified neuronophage. Now we have further characterized this cell type. Following triple immunostaining, we tested a broad panel of mabs (OX-33, OX-19, OX-18, OX-6, R73, ED1, and ED2) to stain, quench fluorescence, and thus immunotype the unknown phagocytes. Only the mab ED2, the classical marker for perivascular cells, specifically stained the small round neuronophages. This surprising migration of perivascular cells toward decaying neurons was additionally tested and confirmed by intracerebroventricular application of FG prior to resection of the facial nerve Providing evidence for neuronophagia by ED2-positive cells, our results strongly support the hypothesis that the latter are the APC (antigen presenting cells) of the CNS.

Animals↗

PubMed1996

Changes in eye blink responses following hypoglossal-facial anastomosis in the cat: evidence of adult mammal motoneuron unadaptability to new motor tasks.

Hypoglossal-facial anastomosis is used in humans to restore the activity of the mimic musculature following irrecoverable facial nerve lesions. As eyelid movement kinetics is very well known, we have used this experimental model in cats to follow the evolution of blink responses and the adaptability of hypoglossal motor pools to new motor tasks. Although the electromyographic activity of the orbicularis oculi muscle in response to corneal air puffs, flashes of light or electrical stimulation of the supraorbital nerve was not recovered in the seven months following this crossed anastomosis, reflex blinks were got back by the increased activity of the retractor bulbi and extraocular recti muscles. The lid of the anastomosed side oscillated in perfect synchronization with tongue movements during licking, while it was severely affected in its motor function during optokinetic stimulation because of the spontaneous appearance of tongue-related hypoglossal activity. Present results suggest that adult mammal motoneurons are unable to readapt their motor programs to the kinetic needs of new motor targets and that most of the functional recovery observed in the cat was achieved by the compensatory hyperactivity of motor systems not directly affected by the surgery.

Adaptation, Physiological↗

PubMed1995

Phagocytic microglia during delayed neuronal loss in the facial nucleus of the rat: time course of the neuronofugal migration of brain macrophages.

The injection of Fluoro-Gold (FG) into the whisker pad of rats yields a stable fluorescent labeling of the motoneurons in the lateral facial subnucleus. Following resection of 8-10 mm of the facial nerve, the microglia phagocytose the FG-preloaded neurons and assume the label. Employing this vital labeling of microglia in situ we studied the fate of same after completion of phagocytic activity. Starting at 56 days post resection (DPR) the FG-labeled microglia spread out from the lateral facial subdivision and invaded the entire facial nucleus. The quantitative analysis of this redistribution of the fluorescent marker revealed a prolonged increase in the number of labeled microglia strictly proportional to the delayed loss of neurons. The differentiation between microglia and shrunken neurons was performed with the new method of immunoquenching: the staining of vibratome sections with anti-rat neuron-specific enolase (NSE) combined with an ABC-HRP kit and DAB as detector totally extinguished (quenched) all fluorescence from the pre-labeled facial motoneurons. The fluorescent microglia were additionally stained with GSA I-B4 and OX-42, which should completely quench all fluorescence in the section. However, a few small round cells, always closely opposed to neuronal perikarya, still fluoresced. These NSE-negative, GSA I-B4 and OX-42 negative, but fluorescent cells may represent a new, immunologically uncharacterized microglial cell type, that participates in neuronophagia.

Animals↗

PubMed1994

Axotomy induces intranuclear immunolocalization of neuron-specific enolase in facial and hypoglossal neurons of the rat.

Neuron-specific enolase as an enzyme of the glycolytic pathway is localized in the cytoplasm of nerve cells, but not in the cell nucleus. We have applied immunocytochemistry with 1:64,000 polyclonal anti-rat neuron-specific enolase to the brainstem of male and female adult Wistar rats following: (a) transection of the facial nerve with immediate microsurgical nerve suture (facial-facial anastomosis), (b) transection of the hypoglossal nerve with immediate suture (hypoglossal-hypoglossal anastomosis) and (c) transection of the facial and hypoglossal nerve with immediate suture of the proximal hypoglossal to the distal facial nerve stump (hypoglossal-facial anastomosis). Studying the intracellular immunolocalization of neuron-specific enolase in neurons of the facial and hypoglossal nucleus we detected that (1) in normal rats about 20% of all facial and hypoglossal neurons display not only cytoplasmic, but also intranuclear neuron-specific enolase-like immunoreactivity and (2) following any axotomy of the facial or hypoglossal peripheral nerve, the perikarya of all injured motoneurons react by an outstanding increase of neuron-specific enolase-like immunoreactivity in the karyoplasm. Similar findings were obtained in experiments on non-fixed cultured Neuro-2a cells that had been lesioned with hydrogen peroxide. Counting the absolute numbers of normal and reactive neurons at 1-365 days post axotomy revealed that the increase of neuron-specific enolase in neuronal cell nuclei is temporary and reversible. It is first detected at 2 days post axotomy, reaches its maximum at 10-18 days post axotomy and is no longer evident 56 days following surgery.(ABSTRACT TRUNCATED AT 250 WORDS)

Anastomosis, Surgical↗

PubMed1993

Recovery of original nerve supply after hypoglossal-facial anastomosis causes permanent motor hyperinnervation of the whisker-pad muscles in the rat.

Hypoglossal-facial anastomosis (HFA), used in humans for the treatment of facial palsy, was experimentally performed in adult female Wistar rats. The time course of facial reinnervation and the extent of the new motor nerve supply of the vibrissal muscles that develops after HFA were estimated by counting all motoneurons in the brainstem labeled by injection of horseradish peroxidase (HRP) into the whisker pad; muscle innervation by motor endplates was not studied. In untreated animals, HRP injection labels 1,254 +/- 54 (mean +/- S.D.; n = 6) motoneurons, localized exclusively in the lateral subdivision of the facial nucleus. Immediately following HFA, this number drops to zero. The first HRP-labeled motoneurons appear in the hypoglossal nucleus at 28 days postoperation (dpo) and at 56 dpo their number reaches 1,096 +/- 48. Unexpectedly, the facial nerve, whose proximal stump has been left as blind end during surgery, additionally sends axons to the facial periphery. This resprouting is first detected at 42 dpo with HRP-marked neurons throughout the facial nucleus lacking somatotopic organization. The number of these labeled neurons also rises with time, and at 56 dpo, a total of 1,797 +/- 142 facial and hypoglossal motoneurons, that is, 43% more motoneurons than in normal animals, supplies the whisker pad. This hyperinnervation, that is, the projection of more motoneurons into the target muscle than under normal conditions--further increases to 1,978 +/- 92 motoneurons at 224 dpo and may provide a new animal model for studying the competitive relationships between motoneurons in their search for peripheral targets.

Anastomosis, Surgical↗

PubMed1992

The hypoglossal-facial anastomosis as model of neuronal plasticity in the rat.

Hypoglossal-facial cross anastomosis (HFA) causes regeneration with change of function, as the axotomized hypoglossal motoneurons sprout into the facial plexus and reinnervate the mimic musculature. Following HFA, hypoglossal-hypoglossal single anastomosis (HHA) and resection of 8-10 mm peripheral hypoglossal nerve in 190 female adult Wistar rats, we compared the axon reactions in the hypoglossal nucleus during 1) regeneration with change of function, 2) regeneration with restoration of original function and 3) degeneration of the nucleus. Following postoperative survival times of 1-16 weeks we estimated the volume of the hypoglossal nucleus and counted the number of hypoglossal neurons with the physical disector on both sides of the brainstem. Additional sections of the same animals were reacted with anti-synaptophysin, anti-GFAP and the isolectin Griffonia simplicifolia I-B4 (GSA I-B4) as cytochemical markers for presynaptic boutons, activated astroglia and microglia. After HHA and HFA all hypoglossal neurons survive and the volume of the hypoglossal nucleus remains constant. Resection of the hypoglossal nerve leads to the loss of one third of the hypoglossal neurons and of one third of the volume of the hypoglossal nucleus within 16 weeks post operation. Hypoglossal-facial anastomosis and hypoglossal-hypoglossal anastomosis differ in postoperative swelling of the hypoglossal nucleus, microglia and astroglia activation and the duration of synaptic stripping. All differences are limited to the acute growth phase during regeneration. It is concluded that hypoglossal-facial anastomosis provides more stimulation and facilitates faster recovery of the hypoglossal nucleus than does hypoglossal-hypoglossal anastomosis.

Anastomosis, Surgical↗

PubMed1990

Distribution of activity of alkaline phosphatase and Mg-dependent adenosine triphosphatase in the cranial dura mater-arachnoid interface zone of the rat.

The distribution of the activity of alkaline phosphatase and Mg-dependent adenosine triphosphatase was studied in the encephalic dura mater-arachnoid borderline (interface) zone of albino Wistar rats. Intense clustering of electron-dense granules that indicated alkaline phosphatase activity was observed in the inner dural cells, the neurothelial cells, the outermost row of the outer arachnoidal cells and in the intercellular cleft between the latter two (the so-called electron-dense band). The remainder of the outer arachnoidal cells contained almost no reaction product. Mg-adenosine triphosphatase activity was distributed differently; a lack of reaction product was observed not only in the outer arachnoidal cells, but also in the zone occupied by the electron-dense band. The data confirm histochemically the barrier properties of the dura mater-arachnoid interface zone.

Alkaline Phosphatase↗

PubMed1990

Ultrastructural characteristics of the cranial dura mater-arachnoid interface layer.

The ultrastructural features of the encephalic dura mater-arachnoid borderline (interface) layer (zone) of rats, rabbits, cats and humans were studied. The rat's interface zone included the electron-lucent epithelium-like arranged fibroblasts of the inner dural layer, the rich in filaments cells of the dural neurothelium, a 20 nm wide intercellular cleft filled with electron-dense material and the dark mitochondria-rich cells of the outer arachnoidal layer; in rabbits and cats, this laminar distinction was less prominent, while in man, it was almost absent.

Animals↗

PubMed1990

Ultrastructural investigation of the meningeal compartment of the blood-cerebrospinal fluid-barrier in rats and cats. A horseradish peroxidase study.

The permeability of the meningeal blood vessels and cellular layers to horseradish peroxidase was studied 5, 10 and 15 minutes following intravasal or intraarachnoidal introduction of the marker. When applied intravasally, the horseradish peroxidase-containing solution easily passed through the walls of all meningeal vessels (dural, pial and the ones traversing the arachnoid space). The cells of the inner dural layer and dural neurotheliun delay the penetration of horseradish peroxidase into the cerebrospinal fluid-filled arachnoid space by 10 min--rats and 15 min--cats. The perivascular leptomeningeal cells and their processes restrict the passage of the marker into the arachnoid space in a similar way. These barrier functions of the leptomeningeal cells and the cells that comprise the interface zone between dura mater and the arachnoid are confirmed by experiments where the marker was injected into the arachnoid space.

Animals↗

PubMed1989

Morphogenesis of rat cranial meninges. A light- and electron-microscopic study.

The meninges of albino Wistar rat embryos, aged between the 11th embryonic day (ED) and birth, were sectioned using a specially constructed device. This technique permits optimal microanatomical preservation of all tissues covering the convexity of the brain: skin, muscle, cartilage or bone, and the meninges. At ED11, the zone situated between the epidermis and the brain is occupied by a mesenchymal network. At ED12, part of this delicate network develops as a dense outer cellular layer, while the remainder retains its reticular appearance, thus forming an inner layer (the future meningeal tissue). At ED13, the dura mater starts to differentiate. At ED14, the bony anlage of the skull can be identified, and along with the proceeding maturation of dura mater some fibrillar structures resembling skeletal muscle fibers appear in the developing arachnoid space. At ED15-17, a primitive interface zone - dura mater/arachnoid - is formed, comprised by an outer electronlucent and an inner electron-dense layer marking the outer aspect of the arachnoidal space. At ED18-19, the innermost cellular row of the inner dural layer transforms into neurothelium, which is separated from the darker arachnoidal cells by an electron-dense band. The arachnoidal trabecular zone with the leptomeningeal cells is formed at ED19. By the end of the prenatal period (ED20-21), its innermost part organizes into an inner arachnoidal layer and an outer and inner pial layer. The results from this study indicate (i) that dura mater and leptomeninges develop from an embryonic network of connective tissue-forming cells, and (ii) that the formation of cerebrospinal fluid (CSF)-containing spaces accompanies the differentiation of the meningeal cellular layers.

Animals↗